CN116829561A - Camptothecin compounds and preparation methods and applications thereof - Google Patents

Abstract

Camptothecins with antitumor activity, and its preparation method and application are provided. In particular to a compound shown in the following or a pharmaceutically acceptable form thereof, and a pharmaceutical composition, a preparation method and application thereof. The compounds are useful as medicaments for the treatment of diseases in which cells proliferate abnormally,

Description

Camptothecin compounds and preparation methods and applications thereof

This application is an application with a CN application number of 202110159956.6 and a filing date of February 5, 2021. The CN application number is 202110533304.4 and an application date of May 17, 2021. The CN application number is 202110718245.8 and the filing date is 2021. Based on the application on June 28, 2021, the CN application number is 202110936768. Priority is claimed and the disclosure of the above-mentioned CN application is hereby incorporated into this application in its entirety.

Technical field

The present invention relates to a class of camptothecin compounds with anti-tumor activity and their conjugates, as well as their preparation methods and applications in the medical field.

Background technique

Camptothecin (CPT, Formula 1) is a five-ring quinoline core compound isolated from Camptotheca acuminata, a plant of the Davidia family. It consists of quinoline ring AB, pyrrole ring C, and pyridone ring D. It consists of α-hydroxylactone ring E, in which the 20-position is in S configuration (see structural formula below). It was introduced into clinical practice in the early 1970s due to its excellent anti-cancer activity. Later, clinical trials were terminated due to severe side effects such as diarrhea and hemorrhagic cystitis.

Research data shows that camptothecin can form a ternary complex with cellular DNA topoisomerase I, thereby inhibiting the unwinding of DNA, causing DNA replication to be blocked, and thus causing cell death (Cancer Res. 1989, 49, 6365). Camptothecin and its derivatives have strong anti-tumor activity in animal models such as lung cancer, breast cancer, colorectal cancer, and ovarian cancer (Nature Review Cancer. 2006, 6, 789).

Currently, multiple camptothecin drugs have been approved for tumor treatment (Med.Res.Rev.2015,35,753). Irinotecan is used for the treatment of colorectal cancer; topotecan is used for the treatment of ovarian cancer; belotecan is used for the treatment of ovarian cancer and small cell lung cancer. Camptothecin derivatives include Exatecan, Rubitecan, Karenitecan, Diflomotecan, Lurtotecan, Gimatecan, Namitecan, Simmitecan, Silatecan, Chimmitecan, Elomotecan, etc.

Camptothecin drugs or derivatives often have blood toxicity caused by bone marrow suppression, such as neutropenia, leukopenia, thrombocytopenia, anemia, etc., as well as gastrointestinal side effects, such as nausea, vomiting, diarrhea, etc. Clinical studies have found that measures to improve the safety and effectiveness of camptothecin compounds include improving their pharmacokinetic properties, regulating activity, reducing dosage, or using their conjugates to form antibody conjugates with antibodies. Therefore, the development of camptothecin compounds and their conjugates with novel structures that can enhance effectiveness and improve safety issues still has high clinical demand and application value.

Contents of the invention

The invention provides novel camptothecin compounds and their conjugates. The camptothecin compounds have good anti-tumor activity and are expected to be used in the treatment of tumor diseases; their conjugates have a wide range of antibody-coupled drug applications. prospect.

A first aspect of the invention provides compounds or pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotopic labels, metabolites and prodrugs thereof, wherein the compound has the following Structure shown:

in,

R ₁ and R ₂ are each independently selected from hydrogen, halogen, C _1-6 alkyl, C _1-6 alkoxy, C _1-6 haloalkyl, hydroxyl, cyano and C _3-6 cycloalkyl; or , R ₁ and R ₂ are connected to adjacent carbon atoms to form a 5-6 membered oxygen-containing heterocyclic ring;

R ₃ is hydrogen or connected to the carbon atom ortho to R ₁ to form a six-membered carbon ring;

A is selected from and one of;

R ₄ is selected from hydrogen, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxyalkyl, C ₃ - ₆ cycloalkyl and 3-6 membered heterocyclyl;

R ₅ and R ₆ are each independently selected from hydrogen, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkylaminoalkyl, C _1-6 alkoxyalkyl, C _2-6 Alkenyl, C _2-6 alkynyl, C _3-6 cycloalkyl, 3-6 membered heterocyclyl, 3-6 membered heterocyclylalkyl, aryl and heteroaryl; or R ₅ and R ₆ are with Adjacent carbon atoms are connected to form a 3-6 membered carbocyclic ring or heterocyclic ring;

m=1 or 2.

In some embodiments, the compound has the structure of Formula (I):

In formula (I), ^Rx is selected from hydrogen, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxyalkyl, C _3-6 cycloalkyl and 3-6 membered heterocycle base;

R ^y and R ^z are not hydrogen at the same time, and are independently selected from hydrogen, C _1-6 alkyl, C _1-6 haloalkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 alkylamine Alkyl group, C _1-6 alkoxyalkyl group, 3-6 membered heterocyclylalkyl group and 3-6 membered heterocyclyl group.

In some embodiments, ^Rx is selected from hydrogen, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxyalkyl, C _3-6 cycloalkyl, and 3-6 membered heterocyclyl ;

R ^y and R ^z are not hydrogen at the same time, and are independently selected from hydrogen, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 alkylaminoalkyl, C _1-6 alkoxyalkyl, 3-6 membered heterocyclylalkyl and 3-6 membered heterocyclyl.

In some embodiments, in formula (I), ^Rx is selected from hydrogen, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxyalkyl, C _3-6 cycloalkyl, 3 ~6-membered heterocyclyl;

R ^y and R ^z are not hydrogen at the same time, and are independently selected from hydrogen, C _2-6 alkenyl, and C _2-6 alkynyl.

In some embodiments, ^Rx is selected from hydrogen or C _1-6 alkyl.

In some embodiments, ^Rx is hydrogen.

In some embodiments, Ry and ^Rz are not simultaneously hydrogen and ^are independently selected from hydrogen, Dimethylaminomethylene, morpholinomethylene and methoxymethylene.

In some embodiments, Ry and ^Rz are not simultaneously hydrogen and ^are independently selected from hydrogen, Dimethylaminomethylene and methoxymethylene.

In some embodiments, R ^y is hydrogen and R ^z is selected from Dimethylaminomethylene, morpholinomethylene and methoxymethylene.

In some embodiments, R ^y is hydrogen and R ^z is selected from Dimethylaminomethylene and methoxymethylene.

In some embodiments, ^Rx is hydrogen, ^Ry is hydrogen, and ^Rz is selected from Dimethylaminomethylene, morpholinomethylene and methoxymethylene.

In some embodiments, ^Rx is hydrogen, ^Ry is hydrogen, and ^Rz is selected from Dimethylaminomethylene and methoxymethylene.

In some embodiments, ^Rx is hydrogen, ^Ry is hydrogen, and ^Rz is selected from and dimethylaminomethylene.

In some embodiments, in Formula (I) everywhere structure.

In some embodiments, in Formula (I) everywhere structure.

In some embodiments, in Formula (I) everywhere structure.

In some embodiments, in Formula (I) everywhere structure.

In some embodiments, in Formula (I) everywhere structure.

In some embodiments, in Formula (I) everywhere structure.

In some embodiments, in Formula (I) everywhere structure.

In some embodiments, in Formula (I) everywhere structure.

In some embodiments, the compound has the structure of Formula (II):

In formula (II), A' is selected from and one of;

R ^x' is selected from hydrogen, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxyalkyl, C ₃ - ₆ cycloalkyl and 3-6 membered heterocyclyl;

R ^y' and R ^z' are independently selected from hydrogen, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxyalkyl, C _1-6 alkylaminoalkyl, C ₃ - ₆ Cycloalkyl, 3-6 membered heterocyclyl, 3-6 membered heterocyclylalkyl, C _2-6 alkenyl, C _2-6 alkynyl, aryl and heteroaryl, or R ^y' and R ^{z '} Connected to adjacent carbon atoms to form a 3-6 membered ring.

In some embodiments, the structure of Formula (II) is as shown in Formula (II)-1 below:

_{In some embodiments ,} ^R _{_} Ring group;

R ^y' and R ^z' are independently selected from hydrogen, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxyalkyl, C _3-6 cycloalkyl, _3-6 membered heterocycle group, C _2-6 alkenyl, C _2-6 alkynyl, aryl, heteroaryl, or R ^y' and R ^z' are connected to adjacent carbon atoms to form a 3-6 membered ring.

In some embodiments, the 3-6 membered ring is selected from 3-6 membered carbocyclic rings or 3-6 membered heterocyclic rings.

In some embodiments, R ^x' is selected from hydrogen and C _1-6 alkyl.

In some embodiments, Rx ^' is selected from hydrogen and methyl.

In some embodiments, Ry ^' and ^Rz' are independently selected from hydrogen, C _1-6 alkyl, C _1-6 alkoxyalkyl, C _1-6 alkylaminoalkyl, C ₃ - ₆ cyclo Alkyl and C _2-6 alkenyl, or R ^y' and R ^z' are connected to adjacent carbon atoms to form a 3-6 membered cycloalkyl group.

In some embodiments, R ^y' is selected from hydrogen and C _1-6 alkyl, R ^z' is selected from hydrogen, C _1-6 alkyl, and C ₃ - ₆ cycloalkyl, or R ^y' and R ^z' Connected to adjacent carbon atoms to form a 3-6 membered ring.

In some embodiments, R ^y' is selected from hydrogen and methyl, R ^z' is selected from hydrogen, methyl, and cyclopropyl, or R ^y' and R ^z' are connected to adjacent carbon atoms to form a 3-membered carbocyclic ring.

In some embodiments, R ^x' is selected from hydrogen and methyl, Ry ^' is selected from hydrogen and methyl, R ^z' is selected from hydrogen, methyl, and cyclopropyl, or Ry ^' and R ^z' are identical The adjacent carbon atoms are connected to form a 3-membered carbon ring.

In some embodiments, R ^x' is hydrogen, Ry ^' is selected from hydrogen and methyl, R ^z' is selected from hydrogen, methyl and cyclopropyl, or Ry ^' and R ^z' are attached to adjacent carbon atoms into a 3-membered carbon ring.

In some embodiments, A' in formula (II) is

In some embodiments, in Formula (II)-1 everywhere structure.

In some embodiments, in Formula (II)-1 everywhere structure.

In some embodiments, in Formula (II)-1 everywhere structure.

In some embodiments, in Formula (II)-1 everywhere structure.

In some embodiments, in Formula (II)-1 everywhere structure.

In some embodiments, in Formula (II)-1 everywhere structure.

In some embodiments, in Formula (II)-1 everywhere structure.

In some embodiments, in Formula (II)-1 everywhere structure.

In some embodiments, the compound has the structure of Formula (III):

In formula (III), A” is selected from and one of;

R ^x ” is selected from hydrogen, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxyalkyl, C ₃ - ₆ cycloalkyl and 3-6 membered heterocyclyl;

R ^y ” and R ^z ” are independently selected from hydrogen, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxyalkyl, C _1-6 alkylaminoalkyl, C _3-6 Cycloalkyl, 3-6 membered heterocyclyl, 3-6 membered heterocyclylalkyl, 4-6 membered heterocyclyl, C _2-6 alkenyl, C _2-6 alkynyl, aryl and heteroaryl , or R ^y ” and R ^z ” are connected to adjacent carbon atoms to form a 3-6 membered ring.

In some embodiments, the structure of the compound of formula (III) is as shown in the following formula (III)-1:

_{In some embodiments ,} ^R _{_} Ring group;

R ^y ” and R ^z ” are independently selected from hydrogen, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxyalkyl, C _3-6 cycloalkyl, _4-6 membered heterocycle group, C _2-6 alkenyl, C _2-6 alkynyl, aryl, heteroaryl, or R ^y ” and R ^z ” are connected to adjacent carbon atoms to form a 3-6 membered ring.

In some embodiments, R ^x ″ is selected from hydrogen and C _1-6 alkyl.

In some embodiments, R ^x ″ is hydrogen.

In some embodiments, the 3-6 membered ring is selected from 3-6 membered carbocyclic rings or 3-6 membered heterocyclic rings.

In some embodiments, R ^y ″ and R ^z ″ are each independently selected from hydrogen, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxyalkyl, C _1-6 alkylamine Alkyl, C _3-6 cycloalkyl and vinyl, or R ^y ″ and R ^z ″ are connected to adjacent carbon _atoms to form a 3-6 membered ring.

In some embodiments, R ^y ″ is hydrogen, R ^z ″ is selected from hydrogen, C _1-6 alkyl, C _3-6 cycloalkyl, and vinyl, or R ^y ″ and R ^z ″ are adjacent carbon atoms _. Connected to form a 3-6 membered carbon ring.

In some embodiments, R ^y ″ is hydrogen, R ^z ″ is selected from hydrogen, methyl, cyclopropyl and vinyl, or R ^{y ″} and R ^z ″ are connected to adjacent carbon atoms to form a 3-membered carbocyclic ring.

^{In some embodiments , R} 3-membered carbon ring.

In some embodiments, A" in formula (III) is

In some embodiments, in Formula (III)-1 everywhere structure.

In some embodiments, in Formula (III)-1 everywhere structure.

In some embodiments, in Formula (III)-1 everywhere structure.

In some embodiments, in Formula (III)-1 everywhere structure.

In some embodiments, in Formula (III)-1 everywhere structure.

In some embodiments, in Formula (III)-1 everywhere structure.

In some embodiments, in Formula (III)-1 everywhere structure.

In some embodiments, in Formula (III)-1 everywhere structure.

In some embodiments, the compound has the structure of Formula (IV):

In formula (IV),

R ^a and R ^b are independently selected from hydrogen, halogen, C _1-6 alkyl, C _1-6 hydroxyalkyl, C _1-6 alkoxyalkyl, C _1-6 alkoxy, C _1-6 haloalkyl , hydroxyl and cyano groups; or R ^a and R ^b are connected to adjacent carbon atoms to form a 5-6 membered oxygen-containing heterocyclic ring;

R ^c and R ^d are independently selected from hydrogen, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxyalkyl, C _1-6 alkylaminoalkyl, C _3-6 cycloalkyl base, 3-6 membered heterocyclyl, 3-6 membered heterocyclylalkyl, C _2-6 alkenyl and C _2-6 alkynyl, or R ^c and R ^d are connected to adjacent carbon atoms to form 3-6 One-membered carbocyclic or heterocyclic rings;

R ^e is selected from hydrogen, C _1-6 alkyl, C _3-6 cycloalkyl, C _1-6 haloalkyl, C _1-6 alkoxyalkyl and C ₂ -C ₅ heterocyclyl;

q=0 or 1;

When q=0, R ^c and R ^d are not hydrogen at the same time.

In some embodiments, R ^a and R ^b are independently selected from hydrogen, halogen, C _1-6 alkyl, C _1-6 alkoxy, C _1-6 haloalkyl, hydroxyl, and cyano; or R ^a and R ^b is connected to adjacent carbon atoms to form a 5-6 membered oxygen-containing heterocyclic ring;

R ^c and R ^d are independently selected from hydrogen, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxyalkyl, C _1-6 alkylaminoalkyl, C _3-6 cycloalkyl base, 3-6 membered heterocyclyl, 3-6 membered heterocyclylalkyl, C _2-6 alkenyl and C _2-6 alkynyl, or R ^c and R ^d are connected to adjacent carbon atoms to form 3-6 One-membered carbocyclic or heterocyclic rings;

R ^e is selected from hydrogen, C _1-6 alkyl, C _3-6 cycloalkyl, C _1-6 haloalkyl, C _1-6 alkoxyalkyl and 4-6 membered heterocyclyl;

q=0 or 1;

When q=0, R ^c and R ^d are not hydrogen at the same time.

In some embodiments, in formula (IV), R ^a and R ^b are independently selected from hydrogen, halogen, C _1-6 alkyl, C _1-6 alkoxy, C _1-6 haloalkyl, hydroxyl, and cyano base; or R ^a and R ^b are connected to adjacent carbon atoms to form a 5-6 membered oxygen-containing heterocyclic ring;

R ^c and R ^d are independently selected from hydrogen, C _1-6 alkyl, C ₃ - ₆ cycloalkyl, C _1-6 haloalkyl, C _1-6 alkoxyalkyl, 4-6 membered heterocyclyl, C _2-6 alkenyl, C _2-6 alkynyl, or R ^c and R ^d are connected to adjacent carbon atoms to form a 3-6 membered carbocyclic ring or heterocyclic ring;

R ^e is selected from hydrogen, C _1-6 alkyl, C ₃ - ₆ cycloalkyl, C _1-6 haloalkyl, C _1-6 alkoxyalkyl, or 4-6 membered heterocyclyl;

q=0 or 1;

When q=0, R ^c and R ^d are not hydrogen at the same time.

In some embodiments, R ^a and R ^b are independently selected from hydrogen, halogen, and C _1-6 alkyl, or R ^a and R ^b are connected to adjacent carbon atoms to form a 5-6 membered oxygen-containing heterocycle.

In some embodiments, R ^a and R ^b are independently selected from hydrogen, fluorine, chlorine, and methyl, or R ^a and R ^b and the benzene ring to which they are attached together form

Where Z is selected from -CH ₂ -, -CD ₂ -, -CH ₂ CH ₂ - and -CF ₂ -.

In some embodiments, R ^a is methyl, R ^b is fluorine, or R ^a and R ^b and the benzene ring to which they are attached together form

In some embodiments, ^Rc and ^Rd are independently selected from hydrogen, C _1-6 alkoxyalkyl and C _1-6 alkylaminoalkyl, or R ^c and R ^d are connected to adjacent carbon atoms to form a 3-6 membered carbocyclic ring.

In some embodiments, ^Rc is hydrogen and ^Rd is selected from hydrogen, Methoxyethyl and cyclopropyl, or R ^c and R ^d are connected to adjacent carbon atoms to form a 3-6 membered carbocyclic ring.

In some embodiments, Re ^is selected from hydrogen and C _1-6 alkyl.

In some embodiments, Re ^is selected from hydrogen and isopropyl.

In some embodiments, R ^a is methyl, R ^b is fluorine, or R ^a and R ^b and the benzene ring to which they are attached together form R ^e is selected from hydrogen and isopropyl, R ^c is hydrogen, R ^d is selected from hydrogen, Methoxyethyl and cyclopropyl, or R ^c and R ^d are connected to adjacent carbon atoms to form a 3-membered carbocyclic ring.

In some embodiments, R ^a is methyl, R ^b is fluorine, or R ^a and R ^b and the benzene ring to which they are attached together form R ^e is selected from hydrogen and isopropyl, R ^c is hydrogen, and R ^d is selected from hydrogen, methoxyethyl and cyclopropyl, or R ^c and R ^d are connected to adjacent carbon atoms to form a 3-membered carbocyclic ring.

In some embodiments, in Formula (IV) everywhere structure.

In some embodiments, in Formula (IV) everywhere structure.

In some embodiments, the compound has the structure of Formula (V):

In formula (V), R is selected from C _3-6 cycloalkyl and C _1-6 alkoxy;

A"'selected from and one of;

^{R _} _{_ _ _ _ _}

R ^y ” ^’ and R ^z ” ^’ are independently selected from hydrogen, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxyalkyl, C _1-6 alkylaminoalkyl, C _{3 -6} cycloalkyl, 3-6 membered heterocyclyl, 3-6 membered heterocyclylalkyl, C _2-6 alkenyl, C _2-6 alkynyl, aryl and heteroaryl, or R ^y ''^' and R ^z ″ ^’ are connected to adjacent carbon atoms to form a 3-6 membered ring.

In some embodiments, R is selected from methoxy and cyclopropyl.

In some embodiments, the structure of the compound of formula (V) is as shown in the following formula (V)-1:

In some embodiments, the 3-6 membered ring is selected from 3-6 membered carbocyclic rings or 3-6 membered heterocyclic rings.

In some embodiments, R ^{y ″} and R ^{z ″} are each independently selected from hydrogen, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxyalkyl, C _1-6 Alkylaminoalkyl, C _3-6 cycloalkyl and vinyl groups, or R ^y "^' and R ^z "^' are connected to adjacent carbon atoms to form a 3-6 membered carbocyclic ring.

In some embodiments, Ry ^{″ ′} and R ^z ″ ^′ are both hydrogen, or Ry ^{″ ′} and R ^z ″ ^′ are connected to adjacent carbon atoms to form a 3-6 membered carbocyclic ring.

In some embodiments, ^Rx "^' is selected from hydrogen and C _1-6 alkyl.

In some embodiments, ^Rx "^' is hydrogen.

In some embodiments, A"' is

In some embodiments, R is selected from methoxy and cyclopropyl, R ^{x ″} is hydrogen, Ry ^{″ and} R ^{z ″} are both hydrogen, or Ry ^{″ and} R ^{z ″} are adjacent to Carbon atoms are connected to form a 3-membered carbon ring.

In some embodiments, in Formula (V)-1 everywhere structure.

In some embodiments, in Formula (V)-1 everywhere structure.

In some embodiments, in Formula (V)-1 everywhere structure.

In some embodiments, in Formula (V)-1 everywhere structure.

In some embodiments, in Formula (V)-1 everywhere structure. In some embodiments, in Formula (V)-1 everywhere structure. In some embodiments, in Formula (V)-1 everywhere structure. In some embodiments, in Formula (V)-1 everywhere structure. In some embodiments, the invention provides the following compounds:

On the other hand, the present invention also provides compounds of formula (VI) or pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotopic labels, metabolites and prodrugs thereof:

M-L-E-D

Formula (VI)

in,

M is the linker site with the antibody or its antigen-binding fragment;

L is the connector connecting connectors M and E;

E is the structural fragment connecting L and D;

D is the structural fragment of a cytotoxic drug.

In some embodiments, M is selected from the following structures:

In some embodiments, M is selected from the following structures:

In some embodiments, L is selected from a divalent structure consisting of one or more of the following: C _1-6 alkylene, -N(R')-, carbonyl, -O-, Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, Val-Cit, Val-Ala, Val-Lys, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), D-Val-Leu- Lys, Gly-Gly-Arg, Ala-Ala-Asn, Ala-Ala-Ala, Val-Lys-Ala, Gly-Gly-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Gly-Gly-Gly,

Wherein R' represents hydrogen, C _1-6 alkyl or alkyl group containing -(CH ₂ CH ₂ O) _r -; r is selected from an integer of 1-10; s is selected from an integer of 1-10.

In some embodiments, L is selected from the following structures:

In some embodiments, L is selected from the following structures:

In some embodiments, E is selected from a single bond, -NH- _CH2- ,

In some embodiments, E is -NH- _CH2- .

In some embodiments, the cytotoxic drug is selected from the group consisting of compounds described in any one of the first aspects of the invention.

In some embodiments, the cytotoxic drug is selected from compounds 1-1 to 1-15; 2-1 to 2-27; 3-1 to 3-26; 4-1 to 4-15 of the invention ; or 5-1 to 5-36.

In some embodiments, D is selected from the structure of a compound described herein after dehydrogenation.

In some embodiments, D is selected from compounds 1-1 to 1-15; 2-1 to 2-27; 3-1 to 3-26; 4-1 to 4-15; or 5- Structure after dehydrogenation from 1 to 5-36.

In some embodiments, D is selected from the following structures:

In some embodiments, D is selected from the following structures:

In some embodiments, M-L-E-D is selected from the following compounds:

In some embodiments, M-L-E-D is selected from the following compounds:

definition

Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to technology as used herein are intended to mean technology as commonly understood in the art, including those variations or equivalent technology that would be apparent to those skilled in the art. Although the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present invention.

The terms "includes," "includes," "has," "contains," or "involves" and their other variations herein are inclusive or open-ended and do not exclude other unrecited elements. or method steps.

As used herein, the "*" marked in the structural formula of a compound indicates that the marked carbon atom is a chiral carbon atom, and the present invention includes a pair of enantiomers formed by the chiral carbon atom. For example, if a compound contains two different chiral carbon atoms, the present invention includes four optical isomers formed by the chiral carbon atoms.

As used in this article, The representation may be stereospecific ((R) or (S)) or non-stereospecific.

The term "alkyl" is defined as a straight or branched saturated aliphatic hydrocarbon radical. In some embodiments, an alkyl group has 1 to 12, such as 1 to 6 carbon atoms. For example, as used herein, the term "C _1-6 alkyl" refers to a linear or branched group of 1 to 6 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl (isobutyl, sec-butyl, tert-butyl, n-pentyl and n-hexyl), which is optionally substituted by 1 or more (such as 1, 2 or 3) suitable substituents.

The term "alkenyl" refers to a straight-chain or branched hydrocarbon group containing at least one carbon-carbon double bond, including, for example, "C _2-6 alkenyl", "C _2-4 alkenyl", etc. Examples include, but are not limited to: vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl Alkenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hexenyl Dialkenyl etc.

The term "alkynyl" refers to a straight or branched chain hydrocarbon group containing at least one carbon-carbon triple bond. Including, for example, "C _2-6 alkynyl", "C _4-6 alkynyl" and the like. Examples include, but are not limited to: ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 1,3-butadiynyl, 1-pentynyl, 2 -Pentynyl, 3-pentynyl, 1,3-pentadiynyl, 1,4-pentadiynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,4 -Hexadiynyl, etc.

The term "cycloalkyl" refers to a saturated cyclic hydrocarbon group, including, but not limited to, monocycloalkyl and bicycloalkyl (such as spirocycloalkyl, pentacycloalkyl and bridged cycloalkyl). The term "C _3-6 cycloalkyl" refers to a cycloalkyl group having 3 to 6 ring-forming carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc., which may be optionally replaced by 1 or Substitution with multiple (such as 1, 2 or 3) suitable substituents, for example methyl substituted cyclopropyl.

The term "carbocycle" or "carbocyclyl" refers to a saturated or partially unsaturated non-aromatic monocyclic or polycyclic structure, a hydrocarbon group connected through ring carbons. Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

The term "carbocycle" refers to a saturated or unsaturated non-aromatic monocyclic or polycyclic (such as bicyclic) hydrocarbon ring (e.g., a monocyclic ring such as a cyclopropane ring, a cyclobutane ring, a cyclopentane ring, a cyclohexane ring, Cycloheptane ring, cyclooctane ring, cyclononane ring, or bicyclic ring, including spiro, fused or bridged systems (such as bicyclo[1.1.1]pentane ring, bicyclo[2.2.1]heptane ring, Bicyclo[3.2.1]octane ring or bicyclo[5.2.0]nonane ring, decalin ring, etc.), which may be optionally substituted by 1 or more (such as 1, 2 or 3) suitable substituents Substitution. The term "3-6 membered carbocyclic ring" refers to a carbocyclic ring containing 3, 4, 5 or 6 ring-forming carbon atoms.

The term "heterocyclyl" or "heterocycle" refers to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic) non-aromatic cyclic structure whose ring atoms are composed of carbon atoms and at least one (e.g. 1, 2 or 3) composed of heteroatoms selected from nitrogen, oxygen and sulfur. A heterocyclyl group can be connected to the rest of the molecule through any ring atom if the valence bond requirements are met. The heterocyclic group in the present invention is preferably a 3-6 membered heterocyclic group. The term "3-6 membered heterocyclyl" used in the present invention refers to a heterocyclic group with 3 to 6 ring atoms, including 3-membered heterocyclyl, 4-membered heterocyclyl, 5-membered heterocyclyl and 6-membered heterocyclyl. Membered heterocyclic groups include nitrogen-containing heterocyclic groups and oxygen-containing heterocyclic groups, such as 4-6-membered heterocyclic groups, such as 4-6-membered nitrogen-containing heterocyclic groups and 4-6-membered oxygen-containing heterocyclic groups. Common heterocyclyl groups include (but are not limited to) azetidinyl, oxetanyl, tetrahydrofuryl, pyrrolidinyl, pyrrolidinonyl, imidazolidinyl, pyrazolidinyl, tetrahydropyranyl, piperidinyl, piperazinyl, morpholinyl. The heterocyclyl group in the present invention may be optionally substituted by one or more substituents described in the present invention. The heterocyclyl group in the present invention is optionally condensed with one or more aromatic or non-aromatic rings.

The term "oxygen-containing heterocycle" refers to the aforementioned heterocycle in which one or more (for example, 1, 2 or 3) ring atoms are oxygen atoms, such as a 5-6 membered oxygen-containing heterocycle. Specific examples include but It is not limited to ethylene oxide ring, tetrahydrofuran ring, furan ring, tetrahydropyran ring, pyran ring, etc. The "nitrogen-containing heterocyclic ring" used in the present invention refers to the heterocyclic ring as described above in which one or more (for example, 1, 2 or 3) ring atoms are nitrogen atoms.

The term "haloalkyl" refers to an alkyl group substituted by one or more (such as 1, 2 or 3) the same or different halogen atoms, where alkyl is as defined above. For example, the term "C _1-6 haloalkyl" as used in the present invention refers to a haloalkyl group having 1 to 6 carbon atoms. Common haloalkyl groups include (but are not limited to) -CH ₂ F, -CHF ₂ , -CF ₃ , -CH ₂ CF ₃ , -CF ₂ CF ₃ , -CH ₂ CH ₂ CF ₃ , -CH ₂ Cl, etc. The haloalkyl groups in the present invention are optionally substituted with one or more substituents described in the present invention.

The term "aryl" refers to a group obtained by removing a hydrogen atom from the carbon atom of the aromatic nucleus of an aromatic hydrocarbon molecule. For example, 6-14 membered aryl groups, specific examples include but are not limited to phenyl, naphthyl, anthracenyl, etc.

The term "heteroaryl" refers to an aromatic cyclic group containing at least one ring member selected from N, O and S. Specific examples include but are not limited to 5-6-membered heteroaryl, 5-6-membered nitrogen-containing heteroaryl, 5-6-membered oxygen-containing heteroaryl, etc., such as furyl, thienyl, pyrrolyl, thiazolyl, isothiazole base, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1, 2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, pyridyl, pyrimidinyl, pyridazine base, pyrazinyl, 1,2,3-triazinyl, 1,3,5-triazinyl, 1,2,4,5-tetrazinyl, etc.

The term "alkoxy" refers to a group having the structure "alkyl-O-", where alkyl is as defined above. For example, C _1-6 alkoxy group, C _1-4 alkoxy group, C _1-3 alkoxy group or C _1-2 alkoxy group, etc. Common alkoxy groups include (but are not limited to) methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentyloxy, hexyloxy Key et al. The alkoxy groups in the present invention are optionally substituted with one or more substituents described in the present invention.

()()The term "alkoxyalkyl" refers to an alkyl group substituted by one or more (eg 1, 2, 3 or 4) alkoxy groups, where alkoxy and alkyl are as defined above . For example, the term "C _1-6 alkoxyalkyl" as used in the present invention refers to an alkoxy group having 1 to 6 carbon atoms substituted by one or more (e.g., 1, 2, 3 or 4) alkoxy groups. of alkyl. Common alkoxyalkyl groups include (but are not limited to) CH ₃ O-CH ₂ -, C ₂ H ₅ -O-CH ₂ -, C ₂ H ₅ -O-CH ₂ CH ₂ -, etc.

The term "halo" or "halogen" group is defined to include F, Cl, Br or I.

The term "nitrogen oxide" refers to an oxide (eg, a mono- or di-oxide) of at least one nitrogen atom in the compound structure of the present application. Nitrogen mono-oxides may exist as a single positional isomer or as a mixture of positional isomers.

The term "substituted" means that one or more (e.g., one, two, three or four) hydrogens on the designated atom are replaced by a selection from the indicated group, provided that no more than the designated atom is present in the case of normal valence and the substitution forms a stable compound. Combinations of substituents and/or variables are permissible only if such combinations form stable compounds.

If a substituent is described as "optionally substituted," the substituent may be (1) unsubstituted or (2) substituted. If a carbon of a substituent is described as optionally substituted with one or more substituents in the substituent list, one or more hydrogens on the carbon (to the extent of any hydrogen present) may be present individually and/or together Replaced by independently selected optional substituents. If the nitrogen of a substituent is described as optionally substituted with one or more of the substituents listed, then one or more hydrogens on the nitrogen (to the extent of any hydrogen present) may each be independently selected as optional. substitution of substituents.

If a substituent is described as being "independently selected from" a group, each substituent is selected independently of the other. Thus, each substituent may be the same as or different from another (other) substituent.

As used herein, the term "one or more" means 1 or more than 1, such as 2, 3, 4, 5 or 10 under reasonable conditions.

Unless otherwise specified, as used herein, the point of attachment of a substituent may be from any suitable position on the substituent.

The term "stereoisomer" means an isomer formed due to at least one asymmetric center. In compounds with one or more (e.g., one, two, three or four) asymmetric centers, they can give rise to racemic mixtures, single enantiomers, diastereomeric mixtures and individual of diastereomers. Certain individual molecules may also exist as geometric isomers (cis/trans). Similarly, compounds of the present invention may exist as mixtures of two or more structurally distinct forms in rapid equilibrium (often referred to as tautomers). Representative examples of tautomers include keto-enol tautomers, phenol-ketone tautomers, nitroso-oxime tautomers, and imine-enamine tautomers. wait. It is understood that the scope of this application encompasses all such products in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%). %) isomers or mixtures thereof.

Solid lines (—) and solid wedges can be used in this article or virtual wedge Draw the carbon-carbon bonds of the compounds of the invention. The use of a solid line to depict a bond to an asymmetric carbon atom is intended to indicate that all possible stereoisomers at that carbon atom are included (eg, a specific enantiomer, a racemic mixture, etc.). The use of solid or imaginary wedges to depict bonds to asymmetric carbon atoms is intended to demonstrate that the stereoisomers shown exist. When present in a racemic mixture, solid and imaginary wedges are used to define relative stereochemistry rather than absolute stereochemistry. Unless otherwise specified, the compounds of the present invention are intended to exist as stereoisomers (which includes cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, They exist in the form of geometric isomers, rotamers, conformational isomers, atropisomers and mixtures thereof). The compounds of the present invention may exhibit more than one type of isomerism and consist of mixtures thereof (eg, racemic mixtures and pairs of diastereoisomers).

The present invention encompasses all possible crystalline forms or polymorphs of the compounds of the invention, which may be a single polymorph or a mixture of more than one polymorph in any proportion.

It will also be understood that certain compounds of the present invention may exist in free form for therapeutic use, or, where appropriate, as pharmaceutically acceptable derivatives thereof. In the present invention, pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable salts, esters, solvates, metabolites or prodrugs that, upon administration to a patient in need thereof, can directly or indirectly Compounds of the invention or metabolites or residues thereof are provided. Therefore, when reference is made herein to "a compound of the invention", it is also intended to encompass the various derivative forms of the compound described above.

Pharmaceutically acceptable salts of the compounds of the present invention include acid addition salts and base addition salts thereof.

Suitable acid addition salts are formed from acids that form pharmaceutically acceptable salts, including aspartate, fumarate, glucoheptonate, gluconate, glucuronate, hexafluorophosphate, and the like.

Suitable base addition salts are formed from bases that form pharmaceutically acceptable salts, including aluminum salts, arginine salts, choline salts, diethylamine salts, and the like.

For a review of suitable salts, see Stahl and Wermuth, "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds of the invention are known to those skilled in the art.

The term "ester" means an ester derived from the compounds of each general formula herein, including physiologically hydrolyzable esters (which can be hydrolyzed under physiological conditions to release the free acid or alcohol form of the compound of the invention). The compounds of the present invention may themselves be esters.

The compounds of the invention may exist in the form of solvates, preferably hydrates, wherein the compounds of the invention comprise as structural elements of the crystal lattice of the compounds a polar solvent, in particular such as water, methanol or ethanol. The amount of polar solvent, especially water, may be present in stoichiometric or non-stoichiometric ratios.

Also included within the scope of the invention are metabolites of the compounds of the invention, ie substances formed in the body upon administration of the compounds of the invention. Such products may result, for example, from oxidation, reduction, hydrolysis, amidation, deamidation, esterification, delipidation, enzymatic hydrolysis, etc. of the administered compound. Accordingly, the invention includes metabolites of the compounds of the invention, including compounds prepared by contacting a compound of the invention with a mammal for a time sufficient to produce metabolites thereof.

The invention further includes within its scope prodrugs of the compounds of the invention. Typically such prodrugs will be functional group derivatives of the compound that are readily converted in vivo to the desired therapeutically active compound. Therefore, in these instances, the term "administering" as used in the treatment methods of the present invention shall include the treatment of various diseases or conditions with prodrug forms of one or more of the claimed compounds, but in The prodrug form is converted in vivo to the compound described above upon administration to an individual. General methods for selecting and preparing suitable prodrug derivatives are described, for example, in "Design of Prodrug", ed. H. Bundgaard, Elsevier, 1985.

The invention further includes within its scope isotopic markers of the compounds of the invention which are identical to the compounds of the invention except that one or more atoms are substituted with the same atomic number but an atomic mass or mass number different from that which predominates in nature Atomic substitution for atomic mass or mass number.

The invention also encompasses compounds of the invention containing protecting groups. In any process for preparing the compounds of the invention, it may be necessary and/or desirable to protect sensitive or reactive groups on any relevant molecules, thereby forming chemically protected forms of the compounds of the invention. This can be achieved by conventional protecting groups, for example, those described in Protective Groups in Organic Chemistry, ed. J.F.W. McOmie, Plenum Press, 1973; and T.W. Greene & P.G.M. Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991 Protecting Groups, these references are incorporated herein by reference. The protecting groups can be removed at an appropriate subsequent stage using methods known in the art.

pharmaceutical composition

In a third aspect, the present invention provides a pharmaceutical composition comprising the compound described in the first or second aspect of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, or solvent thereof compounds, nitrogen oxides, isotope labels, metabolites or prodrugs, and one or more pharmaceutically acceptable carriers.

The term "pharmaceutical composition" refers to a composition that can be used as a medicament and contains a pharmaceutically active ingredient (API) (or therapeutic agent) and optionally one or more pharmaceutically acceptable carriers. The term "pharmaceutically acceptable carrier" refers to an excipient that is administered with a therapeutic agent and that is suitable, within the scope of sound medical judgment, for contact with human and/or other animal tissue without undue toxicity, irritation, Allergic reactions or other problems or complications commensurate with a reasonable benefit/risk ratio.

The above-mentioned pharmaceutical composition can act systemically and/or locally, which can be achieved through suitable dosage forms. The dosage forms include, but are not limited to, tablets, capsules, lozenges, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, and aqueous suspensions. , injectable solutions, elixirs, syrups.

The above pharmaceutical composition may contain 0.01 mg to 1000 mg of at least one compound of the present invention or its pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolism substances or prodrugs.

The present invention also provides a method for preparing the above-mentioned pharmaceutical composition or its corresponding preparation form, which includes at least one compound of the present invention or its pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate substances, nitrogen oxides, isotope labels, metabolites or prodrugs in combination with one or more pharmaceutically acceptable carriers.

Pill box products

In a fourth aspect, the present invention provides a pharmaceutical kit product, which includes:

a) As the first therapeutic agent, at least one compound according to the first or second aspect of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide thereof , isotope markers, metabolites or prodrugs, or the pharmaceutical composition described in the third aspect as the first pharmaceutical composition;

b) the optional presence of at least one further therapeutic agent as a second therapeutic agent, or a pharmaceutical composition comprising an additional therapeutic agent as a second pharmaceutical composition; and

c) Optional packaging and/or instructions.

The above-mentioned kit product may contain 0.01 mg to 1000 mg of at least one compound of the present invention or its pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope markers, metabolism substances or prodrugs.

The present invention also provides a preparation method for the above-mentioned pharmaceutical kit, which includes adding at least one compound of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope thereof A marker, metabolite or prodrug or pharmaceutical composition as described above is combined with the optional presence of at least one other therapeutic agent or a pharmaceutical composition, packaging and/or instructions containing the other therapeutic agent.

medicinal purposes

The compound of the present invention can exhibit a strong effect in inhibiting abnormal cell proliferation.

Therefore, the present application provides compounds of the invention or pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotopic labels, metabolites and prodrugs thereof, or pharmaceutical combinations thereof Drugs used to treat diseases involving abnormal cell proliferation.

In addition, this application also provides the compounds of the present invention or their pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope markers, metabolites and prodrugs or the above-mentioned drugs Use of the composition in the preparation of a medicament for the treatment of diseases involving abnormal cell proliferation.

In some embodiments, the disorder involving abnormal cellular proliferation includes, but is not limited to, tumors, such as advanced solid tumors.

The application also provides compounds of the invention or pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotopic markers, metabolites and prodrugs thereof or pharmaceutical combinations of the invention The use of a substance in the preparation of a preparation for inhibiting the proliferation of tumor cells. In certain embodiments, the formulations are for in vivo or in vitro administration. For example, the formulation can be administered to a subject to inhibit the proliferation of tumor cells in the subject; alternatively, the formulation can be administered to cells in vitro (eg, cell lines or cells from the subject), To inhibit the proliferation of tumor cells in vitro.

Tumors described in the present invention include (but are not limited to): brain tumors, lung cancer, squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrium Cancer, colorectal cancer, liver cancer, kidney cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, prostate cancer, female reproductive tract cancer, carcinoma in situ, lymphoma, neurofibroma, thyroid cancer, bone cancer, skin cancer, brain Cancer, colon cancer, testicular cancer, gastrointestinal stromal tumor, prostate tumor, mast cell tumor, multiple myeloma, melanoma, glioma, or sarcoma.

treatment method

In another aspect, the present invention provides a method for treating diseases related to abnormal cell proliferation, which includes the following steps: adding a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt, ester, or stereoisomer thereof , polymorphs, solvates, nitrogen oxides, isotopic markers, metabolites and prodrugs or the above pharmaceutical compositions are administered to individuals in need thereof.

The term "effective amount" refers to a dose capable of inducing a biological or medical response in a cell, tissue, organ or organism (eg, an individual) and sufficient to achieve the desired preventive and/or therapeutic effect.

Dosage regimens can be adjusted to provide the best desired response. For example, it can be administered in a single dose, in divided doses over time, or in a proportionally reduced or increased dose according to the actual situation. It will be understood that, for any particular individual, specific dosage regimens should be adjusted according to need and the professional judgment of the person administering or supervising the administration of the compositions.

The amount of a compound of the invention administered will depend on the individual circumstances, severity of the disease or condition, rate of administration, disposition of the compound, and the judgment of the prescribing physician. Generally speaking, an effective amount is about 0.001-10000 mg/kg of subject's body weight/day. Where appropriate, the effective amount is about 0.01-1000 mg/kg subject body weight/day. Administration can be from about 0.01 to 1000 mg/kg of subject's body weight every day, every two days, or every three days, and typically from about 0.1 to 500 mg/kg of subject's body weight. Exemplary dosing regimens are once or more daily, or once or more weekly, or once or more monthly. When multiple doses are administered, the intervals between single doses may generally be daily, weekly, monthly, or yearly. Alternatively, administration may be in the form of a sustained-release formulation, in which case less frequent dosing is required. The dosage and frequency of administration may vary based on the half-life of the drug in the subject and may vary depending on whether the application is prophylactic or therapeutic. In prophylactic applications, relatively low doses are administered over a long period of time at relatively infrequent intervals; in therapeutic applications, it is sometimes necessary to administer relatively high doses at shorter intervals until the progression of the disease is retarded or stopped, preferably until the individual Shows partial or complete improvement of disease symptoms, after which prophylactic application can be adopted.

The term "treatment" refers to the alleviation or elimination of a targeted disease or condition. If a subject receives a therapeutic amount of a compound of the present invention or a pharmaceutically acceptable form thereof or a pharmaceutical composition of the present invention, at least one indicator and symptom of the subject is observable and/or detectable. Detected remission and/or improvement indicates that the subject has been successfully "treated." It is understood that treatment includes not only complete treatment, but also less than complete treatment, but achieving some biologically or medically relevant results.

The term "administration/administration/administration" (or "administration") refers to the application of a pharmaceutically active ingredient (such as a compound of the present invention) or a pharmaceutical composition containing a pharmaceutically active ingredient (such as a pharmaceutical composition of the present invention) The process of bringing pharmaceutical active ingredients or pharmaceutical compositions into contact with individuals or their cells, tissues, organs, biological fluids and other parts. Common administration methods include (but are not limited to) oral administration, subcutaneous administration, intramuscular administration, subperitoneal administration, ocular administration, nasal administration, sublingual administration, rectal administration, vaginal administration, etc.

The term "need for" refers to a physician's or other caregiver's judgment that an individual needs or would benefit from a preventive and/or therapeutic procedure, based on the physician's or other caregiver's expertise in the individual's area of expertise. factors.

The term "individual" refers to a human or non-human animal. Individuals of the present invention include individuals suffering from diseases and/or disorders (patients) and normal individuals. Non-human animals of the present invention include all vertebrates, such as non-mammals, such as birds, amphibians, reptiles, etc., and mammals, such as non-human primates, domestic animals and/or domesticated animals (such as sheep, dogs, cats, cows, pigs, etc.).

Preparation

The fourth aspect of the present invention provides a synthesis method of the compound.

The compound of formula (I) in the present invention can be synthesized and prepared by the following synthetic route.

Among them, the meanings of R ^x , R ^y and R ^z are as mentioned above; LG is a leaving group, selected from methanesulfonyl, trifluoromethanesulfonyloxy and halogen, preferably trifluoromethanesulfonyloxy or iodine;

step one:

The compound of formula (I)-IM1 is obtained by a substitution reaction between the compound of formula (I)-SM1 and the compound of formula (I)-SM2.

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, 140°C, preferably 50°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from halogenated hydrocarbons (e.g., dichloromethane (DCM), chloroform (TCM), 1,2-dichloroethane ( 1,2-DCE), etc.), nitriles (such as acetonitrile (AN), etc.), N-methylpyrrolidone (NMP), N,N-dimethylformamide (DMF), N,N-dimethylethane Amide (DMA), tetrahydrofuran (THF), 1,4-dioxane (Dioxane), dimethyl sulfoxide (DMSO) and any combination thereof, preferably acetonitrile.

In some embodiments, this step is carried out in the presence of a suitable base, which includes an organic base or an inorganic base. The organic base can be selected from the group consisting of N,N-diisopropylethylamine (DIPEA), triethylamine (TEA), potassium tert-butoxide (t-BuOK) and pyridine (Py), the inorganic base can be selected from potassium phosphate (K ₃ PO4), sodium hydride (NaH), potassium carbonate (K ₂ CO ₃ ), carbonic acid Sodium (Na ₂ CO ₃ ), sodium bicarbonate (NaHCO ₃ ), cesium carbonate (Cs ₂ CO ₃ ) and NaOH, preferably Na ₂ CO ₃ or NaHCO ₃ ;

Step two:

The compound of formula (I) is obtained by the condensation reaction of the compound of formula (I)-IM1 and the compound of formula (I)-SM3;

In some embodiments, this step is performed under a suitable condensation reagent, which may be selected from HATU, HBTU, EDCI, DCC and HOBT, preferably HATU;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 25°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl Sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably N,N-dimethylformamide.

In some embodiments, this step is carried out in a suitable base, including an organic base or an inorganic base, the organic base can be selected from DIPEA, TEA, t-BuOK and Py, and the inorganic base can be selected from K ₃ PO ₄ , NaH, K ₂ CO ₃ , Na ₂ CO ₃ , Cs ₂ CO ₃ and NaOH, preferably DIPEA.

The compound of formula (II)-1 in the present invention can be synthesized and prepared by the following synthetic route.

Among them, R ^x' , R ^y' and R ^z' have the same meanings as mentioned above; LG is a leaving group, selected from methanesulfonyl, trifluoromethanesulfonyloxy and halogen, preferably trifluoromethanesulfonyloxy Or iodine; PG is a protecting group, selected from

step one

The compound of formula (II)-IM1 is obtained by the substitution reaction of the compound of formula (II)-SM1.

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 50°C, 60°C, 100°C, preferably 50°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, and dimethyl sulfoxide. , n-heptane, n-hexane, ethyl acetate, preferably n-heptane.

Step 2

The compound of formula (II)-IM2 is obtained by the reduction reaction of the compound of formula (II)-IM1;

In some embodiments, this step is carried out in the presence of a suitable reducing agent, which may be selected from palladium catalysts, platinum catalysts, rhodium catalysts, preferably platinum catalysts;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 50°C, 60°C, 100°C, preferably 60°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, and dimethyl sulfoxide. , n-heptane, n-hexane, ethyl acetate, preferably ethyl acetate.

Step 3

The compound of formula (II)-IM3 is obtained through the substitution reaction of the compound of formula (II)-IM2,

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 50°C, 60°C, 100°C, preferably 20°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, and dimethyl sulfoxide. , n-heptane, n-hexane, ethyl acetate, preferably ethyl acetate;

In some embodiments, this step is performed under alkaline conditions, and the reagents that provide alkaline conditions include organic bases and inorganic bases. The organic bases include but are not limited to triethylamine, pyridine, N,N-diiso Propylethylamine, n-butyllithium, lithium diisopropylamide, lithium bistrimethylsilylamide, sodium bistrimethylsilylamide; the inorganic bases include but are not limited to potassium carbonate, sodium carbonate , sodium bicarbonate, potassium tert-butoxide, sodium hydride, sodium hydroxide, potassium hydroxide, preferably triethylamine.

Step 4

The compound of formula (II)-IM4 is obtained through a coupling reaction of the compound of formula (II)-IM3;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 50°C, 60°C, 70°C, 100°C, preferably 70°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, and dimethyl sulfoxide. , n-heptane, n-hexane, ethyl acetate, water, preferably a mixed solvent of tetrahydrofuran and water;

In some embodiments, this step is performed under alkaline conditions, and the reagents that provide alkaline conditions include organic bases and inorganic bases. The organic bases include but are not limited to triethylamine, pyridine, N,N-diiso Propylethylamine, n-butyllithium, lithium diisopropylamide, lithium bistrimethylsilylamide, sodium bistrimethylsilylamide; the inorganic bases include but are not limited to potassium carbonate, sodium carbonate , sodium bicarbonate, potassium tert-butoxide, sodium hydride, sodium hydroxide, potassium hydroxide, preferably N,N-diisopropylethylamine.

Step 5

The compound of formula (II)-IM5 is obtained by the reduction reaction of the compound of formula (II)-IM4;

In some embodiments, this step is performed under a suitable reducing agent, which may be selected from palladium catalysts, platinum catalysts, rhodium catalysts, preferably platinum catalysts;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 40°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, and dimethyl sulfoxide. , n-heptane, n-hexane, ethyl acetate, preferably tetrahydrofuran.

Step 6

Compounds of formula (II)-IM6 are obtained through ring-closing reactions of compounds of formula (II)-IM5;

In some embodiments, this step is performed at a suitable temperature, which is 5°C, 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 5°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of trifluoroacetic acid, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, Methyl sulfoxide, n-heptane, n-hexane, ethyl acetate, tert-butanol, preferably a mixed solvent of tetrahydrofuran and tert-butanol.

Step 7

The compound of formula (II)-IM7 is obtained by the substitution reaction of the compound of formula (II)-IM6;

In some embodiments, this step is performed at a suitable temperature, which is 5°C, 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 5°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of trifluoroacetic acid, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, Methyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably trifluoroacetic acid;

In some embodiments, this step is performed under alkaline conditions, and the reagents that provide alkaline conditions include organic bases and inorganic bases. The organic bases include but are not limited to triethylamine, pyridine, N,N-diiso Propylethylamine, n-butyllithium, lithium diisopropylamide, lithium bistrimethylsilylamide, sodium bistrimethylsilylamide; the inorganic bases include but are not limited to potassium carbonate, sodium carbonate , sodium bicarbonate, potassium tert-butoxide, sodium hydride, sodium hydroxide, potassium hydroxide, preferably potassium tert-butoxide.

Step 8

The compound of formula (II)-IM8 is obtained by the reduction reaction of the compound of formula (II)-IM7;

In some embodiments, this step is performed under a suitable reducing agent, which may be selected from palladium catalysts, platinum catalysts, rhodium catalysts, preferably palladium catalysts;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 20°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl Sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably methanol.

Step 9

The compound of formula (II)-IM9 is obtained through the substitution reaction of the compound of formula (II)-IM8;

In some embodiments, this step is performed at a suitable temperature, which is 5°C, 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 20°C;

In some embodiments, this step is performed under alkaline conditions, and the reagents that provide alkaline conditions include organic bases and inorganic bases. The organic bases include but are not limited to triethylamine, pyridine, N,N-diiso Propylethylamine, n-butyllithium, lithium diisopropylamide, lithium bistrimethylsilylamide, sodium bistrimethylsilylamide; the inorganic bases include but are not limited to potassium carbonate, sodium carbonate , sodium bicarbonate, potassium tert-butoxide, sodium hydride, sodium hydroxide, potassium hydroxide, preferably pyridine.

Step 10

The compound of formula (II)-IM10 is obtained through a hydrolysis reaction of the compound of formula (II)-IM9;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 60°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl Sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably methanol.

In some embodiments, the reaction is carried out under acidic conditions, and the reagent providing acidic conditions includes hydrochloric acid, trifluoroacetic acid, formic acid, sulfuric acid, methanesulfonic acid, preferably hydrochloric acid.

Step 11

By compound of formula (II)-IM10 and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyran O[3,4-F]indolazine-3,6,10 (4H)-ketone undergoes cyclization reaction to obtain compound of formula (II)-IM11;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, 140°C, preferably 140°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of toluene, methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, Methyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably toluene.

In some embodiments, the reaction is carried out under acidic conditions, and the reagent providing acidic conditions includes p-toluenesulfonic acid, hydrochloric acid, trifluoroacetic acid, formic acid, sulfuric acid, methanesulfonic acid, preferably p-toluenesulfonic acid.

Step 12

The compound of formula (II)-IM12 is obtained through a hydrolysis reaction of the compound of formula (II)-IM11;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, 140°C, preferably 100°C;

In some embodiments, the reaction is carried out under acidic conditions, and the reagent providing acidic conditions includes p-toluenesulfonic acid, hydrochloric acid, trifluoroacetic acid, formic acid, sulfuric acid, methanesulfonic acid, preferably hydrochloric acid.

Step Thirteen

The compound of formula (II)-IM13 is obtained by substitution reaction between the compound of formula (II)-IM12 and the compound of formula (II)-SM2.

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, 140°C, preferably 50°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from halogenated hydrocarbons (e.g., dichloromethane (DCM), chloroform (TCM), 1,2-dichloroethane ( 1,2-DCE), etc.), nitriles (such as acetonitrile (AN), etc.), N-methylpyrrolidone (NMP), N,N-dimethylformamide (DMF), N,N-dimethylethane Amide (DMA), tetrahydrofuran (THF), 1,4-dioxane (Diox), dimethyl sulfoxide (DMSO) and any combination thereof, preferably acetonitrile.

In some embodiments, this step is carried out in the presence of a suitable base, which includes an organic base or an inorganic base. The organic base can be selected from the group consisting of N,N-diisopropylethylamine (DIPEA), triethylamine (TEA), potassium tert-butoxide (t-BuOK) and pyridine (Py), the inorganic base can be selected from potassium phosphate (K ₃ PO ₄ ), sodium hydride (NaH), potassium carbonate (K ₂ CO ₃ ), Sodium carbonate (Na ₂ CO ₃ ), sodium bicarbonate (NaHCO ₃ ), cesium carbonate (Cs ₂ CO ₃ ) and NaOH, preferably Na ₂ CO ₃ or NaHCO ₃ ;

Step 14

The compound of formula (II)-IM14 is obtained by the condensation reaction of the compound of formula (II)-IM13 and the compound of formula (II)-SM3;

In some embodiments, this step is performed under a suitable condensation reagent, which may be selected from HATU, HBTU, EDCI, DCC and HOBT, preferably HATU;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 25°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl Sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably N,N-dimethylformamide.

In some embodiments, this step is carried out in a suitable base, including an organic base or an inorganic base, the organic base can be selected from DIPEA, TEA, t-BuOK and Py, and the inorganic base can be selected from K ₃ PO ₄ , NaH, K ₂ CO ₃ , Na ₂ CO ₃ , Cs ₂ CO ₃ and NaOH, preferably DIPEA.

Step 15

The compound of formula (II)-1 is obtained through the acidolysis reaction of the compound of formula (II)-IM14;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, 140°C, preferably 25°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl Sulfoxide, n-heptane, n-hexane, ethyl acetate and their mixed solvents, preferably a mixed solution of methylene chloride and methanol (volume ratio 2:1).

In some embodiments, the reaction is carried out under acidic conditions, and the reagent providing acidic conditions includes p-toluenesulfonic acid, hydrochloric acid, trifluoroacetic acid, formic acid, sulfuric acid, methanesulfonic acid, preferably hydrochloric acid.

The synthesis method of formula (II)-SM3 is as follows:

When PG is hour:

step one

The compound of formula (II)-SM3-3 is obtained by the substitution reaction of the compound of formula (II)-SM3-1 and the compound of formula (II)-SM3-2;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, 140°C, preferably 0-25°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl Sulfoxide, n-heptane, n-hexane, ethyl acetate and their mixed solvents, preferably tetrahydrofuran.

In some embodiments, this step is carried out in a suitable base, including an organic base or an inorganic base, the organic base can be selected from DIPEA, TEA, t-BuOK and Py, and the inorganic base can be selected from K ₃ PO ₄ , NaH, K ₂ CO ₃ , Na ₂ CO ₃ , Cs ₂ CO ₃ and NaOH, preferably K ₂ CO ₃ .

Step 2

The compound of formula (II)-SM3 is obtained by hydrogenation reaction of the compound of formula (II)-SM3-3;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, 140°C, preferably 25°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl Sulfoxide, n-heptane, n-hexane, ethyl acetate and their mixed solvents, preferably methanol.

In some embodiments, this step is performed under a suitable reducing agent, which may be selected from palladium catalysts, platinum catalysts, rhodium catalysts, preferably palladium catalysts;

When PG is When , R ^y' and R ^z' are hydrogen:

step one

The compound of formula (II)-SM3 is obtained by the condensation reaction of the compound of formula (II)-SM3-4 and the compound of formula (II)-SM3-5;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, 140°C, preferably 25°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl Sulfoxide, n-heptane, n-hexane, ethyl acetate and their mixed solvents, preferably N,N-dimethylformamide.

Alternatively, the compound of formula (II)-1 can be synthesized and prepared by the following synthetic route:

step one

The compound of formula (II)-IM15 is obtained through the condensation reaction of the compound of formula (II)-IM13 and the compound of formula (II)-SM4;

In some embodiments, this step is performed under a suitable condensation reagent, which may be selected from HATU, HBTU, EDCI, DCC and HOBT, preferably HATU;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 25°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl Sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably N,N-dimethylformamide.

In some embodiments, this step is carried out in a suitable base, including an organic base or an inorganic base, the organic base can be selected from DIPEA, TEA, t-BuOK and Py, and the inorganic base can be selected from K ₃ PO ₄ , NaH, K ₂ CO ₃ , Na ₂ CO ₃ , Cs ₂ CO ₃ and NaOH, preferably DIPEA.

Step 2

The compound of formula (II)-1 is obtained by removing the silicon protecting group from the compound of formula (II)-IM15;

Alternatively, the compound of formula (II) can be synthesized and prepared by the following synthetic route:

step one

The compound of formula (II) is obtained by the condensation reaction of the compound of formula (II)-IM13 and the compound of formula (II)-SM5;

In some embodiments, this step is performed under a suitable condensation reagent, which may be selected from the group consisting of HATU, HBTU, EDCI, DCC and HOBT, preferably HATU;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 25°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl Sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably N,N-dimethylformamide.

In some embodiments, this step is carried out in a suitable base, including an organic base or an inorganic base, the organic base can be selected from DIPEA, TEA, t-BuOK and Py, and the inorganic base can be selected from K ₃ PO ₄ , NaH, K ₂ CO ₃ , Na ₂ CO ₃ , Cs ₂ CO ₃ and NaOH, preferably DIPEA.

The compound of formula (III)-1 in the present invention can be synthesized and prepared by the following synthetic route.

Among them, R ^x ”, R ^y ” and R ^z ” have the same meanings as mentioned above; LG is a leaving group, selected from methanesulfonyl, trifluoromethanesulfonyloxy and halogen, preferably trifluoromethanesulfonyloxy Or chlorine; PG is a protecting group, selected from

step one

The compound of formula (III)-IM1 is obtained by the substitution reaction of the compound of formula (III)-SM1.

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 50°C, 60°C, 100°C, preferably 50°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, and dimethyl sulfoxide. , n-heptane, n-hexane, ethyl acetate, preferably n-heptane.

Step 2

The compound of formula (III)-IM2 is obtained by the reduction reaction of the compound of formula (III)-IM1;

In some embodiments, this step is carried out in the presence of a suitable reducing agent, which may be selected from palladium catalysts, platinum catalysts, rhodium catalysts, preferably platinum catalysts;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 50°C, 60°C, 100°C, preferably 60°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, and dimethyl sulfoxide. , n-heptane, n-hexane, ethyl acetate, preferably ethyl acetate.

Step 3

The compound of formula (III)-IM3 is obtained through the substitution reaction of the compound of formula (III)-IM2,

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 50°C, 60°C, 100°C, preferably 20°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, and dimethyl sulfoxide. , n-heptane, n-hexane, ethyl acetate, preferably ethyl acetate;

In some embodiments, this step is performed under alkaline conditions, and the reagents that provide alkaline conditions include organic bases and inorganic bases. The organic bases include but are not limited to triethylamine, pyridine, N,N-diiso Propylethylamine, n-butyllithium, lithium diisopropylamide, lithium bistrimethylsilylamide, sodium bistrimethylsilylamide; the inorganic bases include but are not limited to potassium carbonate, sodium carbonate , sodium bicarbonate, potassium tert-butoxide, sodium hydride, sodium hydroxide, potassium hydroxide, preferably triethylamine.

Step 4

The compound of formula (III)-IM4 is obtained through a coupling reaction of the compound of formula (III)-IM3;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 50°C, 60°C, 70°C, 100°C, preferably 70°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, and dimethyl sulfoxide. , n-heptane, n-hexane, ethyl acetate, water, preferably a mixed solvent of tetrahydrofuran and water;

In some embodiments, this step is performed under alkaline conditions, and the reagents that provide alkaline conditions include organic bases and inorganic bases. The organic bases include but are not limited to triethylamine, pyridine, N,N-diiso Propylethylamine, n-butyllithium, lithium diisopropylamide, lithium bistrimethylsilylamide, sodium bistrimethylsilylamide; the inorganic bases include but are not limited to potassium carbonate, sodium carbonate , sodium bicarbonate, potassium tert-butoxide, sodium hydride, sodium hydroxide, potassium hydroxide, preferably N,N-diisopropylethylamine.

Step 5

The compound of formula (III)-IM5 is obtained by the reduction reaction of the compound of formula (III)-IM4;

In some embodiments, this step is performed under a suitable reducing agent, which may be selected from palladium catalysts, platinum catalysts, rhodium catalysts, preferably platinum catalysts;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 40°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, and dimethyl sulfoxide. , n-heptane, n-hexane, ethyl acetate, preferably tetrahydrofuran.

Step 6

The compound of formula (III)-IM6 is obtained through the ring-closing reaction of the compound of formula (III)-IM5;

In some embodiments, this step is performed at a suitable temperature, which is 5°C, 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 5°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of trifluoroacetic acid, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, Methyl sulfoxide, n-heptane, n-hexane, ethyl acetate, tert-butanol, preferably a mixed solvent of tetrahydrofuran and tert-butanol.

Step 7

The compound of formula (III)-IM7 is obtained through the substitution reaction of the compound of formula (III)-IM6;

In some embodiments, this step is performed at a suitable temperature, which is 5°C, 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 5°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of trifluoroacetic acid, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, Methyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably trifluoroacetic acid;

In some embodiments, this step is performed under alkaline conditions, and the reagents that provide alkaline conditions include organic bases and inorganic bases. The organic bases include but are not limited to triethylamine, pyridine, N,N-diiso Propylethylamine, n-butyllithium, lithium diisopropylamide, lithium bistrimethylsilylamide, sodium bistrimethylsilylamide; the inorganic bases include but are not limited to potassium carbonate, sodium carbonate , sodium bicarbonate, potassium tert-butoxide, sodium hydride, sodium hydroxide, potassium hydroxide, preferably potassium tert-butoxide.

Step 8

The compound of formula (III)-IM8 is obtained by the reduction reaction of the compound of formula (III)-IM7;

In some embodiments, this step is performed under a suitable reducing agent, which may be selected from palladium catalysts, platinum catalysts, rhodium catalysts, preferably palladium catalysts;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 20°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl Sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably methanol.

Step 9

The compound of formula (III)-IM9 is obtained through the substitution reaction of the compound of formula (III)-IM8;

In some embodiments, this step is performed at a suitable temperature, which is 5°C, 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 20°C;

In some embodiments, this step is performed under alkaline conditions, and the reagents that provide alkaline conditions include organic bases and inorganic bases. The organic bases include but are not limited to triethylamine, pyridine, N,N-diiso Propylethylamine, n-butyllithium, lithium diisopropylamide, lithium bistrimethylsilylamide, sodium bistrimethylsilylamide; the inorganic bases include but are not limited to potassium carbonate, sodium carbonate , sodium bicarbonate, potassium tert-butoxide, sodium hydride, sodium hydroxide, potassium hydroxide, preferably pyridine.

Step 10

The compound of formula (III)-IM10 is obtained through the hydrolysis reaction of the compound of formula (III)-IM9;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 60°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl Sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably methanol.

In some embodiments, the reaction is carried out under acidic conditions, and the reagent providing acidic conditions includes hydrochloric acid, trifluoroacetic acid, formic acid, sulfuric acid, methanesulfonic acid, preferably hydrochloric acid.

Step 11

By compound of formula (III)-IM10 and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyran O[3,4-F]indolazine-3,6,10 (4H)-ketone undergoes cyclization reaction to obtain compound of formula (III)-IM11;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, 140°C, preferably 140°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of toluene, methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, Methyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably toluene.

In some embodiments, the reaction is carried out under acidic conditions, and the reagent providing acidic conditions includes p-toluenesulfonic acid, hydrochloric acid, trifluoroacetic acid, formic acid, sulfuric acid, methanesulfonic acid, preferably p-toluenesulfonic acid.

Step 12

The compound of formula (III)-IM12 is obtained through a hydrolysis reaction of the compound of formula (III)-IM11;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, 140°C, preferably 100°C;

In some embodiments, the reaction is carried out under acidic conditions, and the reagent providing acidic conditions includes p-toluenesulfonic acid, hydrochloric acid, trifluoroacetic acid, formic acid, sulfuric acid, methanesulfonic acid, preferably hydrochloric acid.

Step Thirteen

The compound of formula (III)-IM13 is obtained by substitution reaction between the compound of formula (III)-IM12 and the compound of formula (III)-SM2.

A substitution reaction occurs to obtain the compound of formula (II)-IM13.

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, 140°C, preferably 50°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from halogenated hydrocarbons (e.g., dichloromethane (DCM), chloroform (TCM), 1,2-dichloroethane ( 1,2-DCE), etc.), nitriles (such as acetonitrile (AN), etc.), N-methylpyrrolidone (NMP), N,N-dimethylformamide (DMF), N,N-dimethylethane Amide (DMA), tetrahydrofuran (THF), 1,4-dioxane (Dioxane), dimethyl sulfoxide (DMSO) and any combination thereof, preferably acetonitrile.

In some embodiments, this step is carried out in the presence of a suitable base, which includes an organic base or an inorganic base. The organic base can be selected from the group consisting of N,N-diisopropylethylamine (DIPEA), triethylamine (TEA), potassium tert-butoxide (t-BuOK) and pyridine (Py), the inorganic base can be selected from potassium phosphate (K ₃ PO ₄ ), sodium hydride (NaH), potassium carbonate (K ₂ CO ₃ ), Sodium carbonate (Na ₂ CO ₃ ), sodium bicarbonate (NaHCO ₃ ), cesium carbonate (Cs ₂ CO ₃ ) and NaOH, preferably Na ₂ CO ₃ or NaHCO ₃ ;

Step 14

The compound of formula (III)-IM14 is obtained by the condensation reaction of the compound of formula (III)-IM13 and the compound of formula (III)-SM3;

In some embodiments, this step is performed under a suitable condensation reagent, which may be selected from HATU, HBTU, EDCI, DCC and HOBT, preferably HATU;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 25°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl Sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably N,N-dimethylformamide.

In some embodiments, this step is carried out in a suitable base, including an organic base or an inorganic base, the organic base can be selected from DIPEA, TEA, t-BuOK and Py, and the inorganic base can be selected from K ₃ PO ₄ , NaH, K ₂ CO ₃ , Na ₂ CO ₃ , Cs ₂ CO ₃ and NaOH, preferably DIPEA.

Step 15

The compound of formula (III)-1 is obtained through an acidolysis reaction of the compound of formula (III)-IM14;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, 140°C, preferably 25°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl Sulfoxide, n-heptane, n-hexane, ethyl acetate and their mixed solvents, preferably a mixed solution of methylene chloride and methanol (volume ratio 2:1).

In some embodiments, the reaction is carried out under acidic conditions, and the reagent providing acidic conditions includes p-toluenesulfonic acid, hydrochloric acid, trifluoroacetic acid, formic acid, sulfuric acid, methanesulfonic acid, preferably hydrochloric acid.

The synthesis method of formula (III)-SM3 is as follows:

When PG is hour:

step one:

The compound of formula (III)-SM3-3 is obtained through the substitution reaction of the compound of formula (III)-SM3-1 and the compound of formula (II)-SM3-2;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, 140°C, preferably 0-25°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl Sulfoxide, n-heptane, n-hexane, ethyl acetate and their mixed solvents, preferably tetrahydrofuran.

In some embodiments, this step is carried out in a suitable base, including an organic base or an inorganic base, the organic base can be selected from DIPEA, TEA, t-BuOK and Py, and the inorganic base can be selected from K ₃ PO ₄ , NaH, K ₂ CO ₃ , Na ₂ CO ₃ , Cs ₂ CO ₃ and NaOH, preferably K ₂ CO ₃ .

Step 2:

The compound of formula (III)-SM3 is obtained by hydrogenation reaction of the compound of formula (III)-SM3-3;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, 140°C, preferably 25°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl Sulfoxide, n-heptane, n-hexane, ethyl acetate and their mixed solvents, preferably methanol.

In some embodiments, this step is performed under a suitable reducing agent, which may be selected from palladium catalysts, platinum catalysts, rhodium catalysts, preferably palladium catalysts;

When PG is When , R ^y' and R ^z' are hydrogen:

step one:

The compound of formula (III)-SM3 is obtained by the condensation reaction of the compound of formula (III)-SM3-4 and the compound of formula (III)-SM3-5;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, 140°C, preferably 25°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl Sulfoxide, n-heptane, n-hexane, ethyl acetate and their mixed solvents, preferably N,N-dimethylformamide.

Alternatively, the compound of formula (III)-1 can be synthesized and prepared by the following synthetic route:

step one:

The compound of formula (III)-IM15 is obtained by the condensation reaction of the compound of formula (III)-IM13 and the compound of formula (III)-SM4;

In some embodiments, this step is performed under a suitable condensation reagent, which may be selected from HATU, HBTU, EDCI, DCC and HOBT, preferably HATU;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 25°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl Sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably N,N-dimethylformamide.

In some embodiments, this step is carried out in a suitable base, including an organic base or an inorganic base, the organic base can be selected from DIPEA, TEA, t-BuOK and Py, and the inorganic base can be selected from K ₃ PO ₄ , NaH, K ₂ CO ₃ , Na ₂ CO ₃ , Cs ₂ CO ₃ and NaOH, preferably DIPEA.

Step 2:

The compound of formula (III)-1 is obtained by removing the silicon protecting group from the compound of formula (III)-IM15;

Alternatively, the compound of formula (III)-1 can be synthesized and prepared by the following synthetic route:

step one:

The compound of formula (III)-1 is obtained through the condensation reaction of the compound of formula (III)-IM13 and the compound of formula (III)-SM5;

In some embodiments, this step is performed under a suitable condensation reagent, which may be selected from HATU, HBTU, EDCI, DCC and HOBT, preferably HATU;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 25°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl Sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably N,N-dimethylformamide.

In some embodiments, this step is carried out in a suitable base, including an organic base or an inorganic base, the organic base can be selected from DIPEA, TEA, t-BuOK and Py, and the inorganic base can be selected from K ₃ PO ₄ , NaH, K ₂ CO ₃ , Na ₂ CO ₃ , Cs ₂ CO ₃ and NaOH, preferably DIPEA.

The compound of formula (IV) in the present invention can be synthesized and prepared by the following synthetic route:

Among them, R ^a , R ^b , R ^c , R ^d and R ^e have the same meanings as mentioned above;

When q=1,

step one

The compound of formula (IV)-IM1 is obtained through the nitration reaction of the compound of formula (IV)-SM1;

In some embodiments, this step is performed at a suitable temperature of 5°C, 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 25°C.

Step 2

The compound of formula (IV)-IM2 is obtained by hydrogenation reaction of the compound of formula (IV)-IM1;

In some embodiments, this step is performed under a suitable reducing agent, which may be selected from palladium catalysts, platinum catalysts, rhodium catalysts, preferably palladium catalysts;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 25°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl Sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably ethyl acetate.

Step 3

The compound of formula (IV)-IM3 is obtained through the acylation reaction of the compound of formula (IV)-IM2,

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 50°C, 60°C, 100°C, preferably 25°C;

Step 4

The compound of formula (IV)-IM4 is obtained by reacting the compound of formula (IV)-IM3 with DMF-DMA;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, 120°C, preferably 120°C;

Step 5

The compound of formula (IV)-IM5 is obtained through the substitution reaction of the compound of formula (IV)-IM4 and the compound of formula (IV)-SM2;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, 140°C, preferably 50°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from methanol, ethanol, N, N-methylpyrrolidone, dimethyl sulfoxide, preferably ethanol;

Step 6

The compound of formula (IV)-IM6 is obtained by the reduction reaction of the compound of formula (IV)-IM5;

In some embodiments, this step is performed in the presence of a suitable reducing agent, preferably sodium borohydride;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 0-25°C;

In some embodiments, this step is carried out in a suitable organic solvent, which may be selected from the group consisting of tetrahydrofuran, glacial acetic acid, methanol and mixed solutions thereof, preferably glacial acetic acid.

Step 7

The compound of formula (IV)-IM7 is obtained by protecting the amino group of the compound of formula (IV)-IM6 with Fmoc;

Step 8

The compound of formula (IV)-IM8 is obtained by removing the acetyl protecting group from the amino group of the compound of formula (IV)-IM7;

Step 9

The compound of formula (IV)-IM9 is obtained by ring-closing reaction of the compound of formula (IV)-IM8 under acidic conditions;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, 120°C, preferably 120°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from toluene, xylene, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, Toluene and xylene are preferred;

In some embodiments, this step is performed under acidic conditions;

Reagents providing acidic conditions include p-toluenesulfonic acid, hydrochloric acid, trifluoroacetic acid, formic acid, sulfuric acid, methanesulfonic acid, preferably p-toluenesulfonic acid.

Step 10

The compound of formula (IV)-IM10 is obtained by removing the Fmoc protecting group from the compound of formula (IV)-IM9;

Step 11

The compound of formula (IV) is obtained by the condensation reaction of the compound of formula (IV)-IM10 and the compound of formula (IV)-SM4;

In some embodiments, this step is performed under a suitable condensation reagent, which may be selected from the group consisting of HATU, HBTU, EDCI, DCC and HOBT, preferably HBTU;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 25°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl Sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably N,N-dimethylformamide.

In some embodiments, this step is carried out in a suitable base, including an organic base or an inorganic base, the organic base can be selected from DIPEA, TEA, t-BuOK and Py, and the inorganic base can be selected from K ₃ PO ₄ , NaH, K ₂ CO ₃ , Na ₂ CO ₃ , Cs ₂ CO ₃ and NaOH, preferably DIPEA.

Or, when q=0,

LG is a leaving group, selected from methanesulfonyl, trifluoromethanesulfonyloxy and halogen, preferably trifluoromethanesulfonyloxy or iodine;

step one

The compound of formula (IV)-IM11 is obtained by the reduction reaction of the compound of formula (IV)-SM5,

In some embodiments, this step is carried out in the presence of a suitable reducing agent, which may be selected from palladium catalysts, platinum catalysts, rhodium catalysts, preferably platinum catalysts;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 50°C, 60°C, 100°C, preferably 25°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, and dimethyl sulfoxide. , n-heptane, n-hexane, ethyl acetate, preferably ethyl acetate and tetrahydrofuran.

Step 2

The compound of formula (IV)-IM12 is obtained by Friedel-Crafts acylation reaction of the compound of formula (IV)-IM11;

In some embodiments, this step is performed at a suitable temperature of 5°C, 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 25°C.

Step 3

The compound of formula (IV)-IM13 is obtained by ring-closing reaction of formula (IV)-IM12 under acidic conditions;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, 120°C, preferably 120°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from toluene, xylene, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, Toluene and xylene are preferred;

In some embodiments, this step is performed under acidic conditions;

Reagents providing acidic conditions include p-toluenesulfonic acid, hydrochloric acid, trifluoroacetic acid, formic acid, sulfuric acid, methanesulfonic acid, preferably p-toluenesulfonic acid.

Step 4

The compound of formula (IV)-IM14 is obtained through the substitution reaction of formula (IV)-IM13;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, 140°C, preferably 25°C;

In some embodiments, this step is carried out in a suitable organic solvent, which may be selected from methanol, ethanol, N, N-methylpyrrolidone, dimethyl sulfoxide, preferably dimethyl sulfoxide;

Step 5

The compound of formula (IV)-IM15 is obtained by the reduction reaction of the compound of formula (IV)-IM14,

In some embodiments, this step is carried out in the presence of a suitable reducing agent, which may be selected from palladium catalysts, platinum catalysts, rhodium catalysts, triphenylphosphine, triethyl phosphite, preferably triethyl phosphite ;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 50°C, 60°C, 80°C, 100°C, preferably 80°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl Sulfoxide, n-heptane, n-hexane, ethyl acetate, toluene, and mixed solutions thereof, preferably a mixed solution of methanol and toluene.

Step 6

The compound of formula (IV)-IM16 is obtained by the substitution reaction between the compound of formula (IV)-IM15 and the compound of formula (IV)-SM6.

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, 140°C, preferably 50°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from halogenated hydrocarbons (e.g., dichloromethane (DCM), chloroform (TCM), 1,2-dichloroethane ( 1,2-DCE), etc.), nitriles (such as acetonitrile (AN), etc.), N-methylpyrrolidone (NMP), N,N-dimethylformamide (DMF), N,N-dimethylethane Amide (DMA), tetrahydrofuran (THF), 1,4-dioxane (Dioxane), dimethyl sulfoxide (DMSO) and any combination thereof, preferably acetonitrile.

In some embodiments, this step is carried out in the presence of a suitable base, which includes an organic base or an inorganic base. The organic base can be selected from the group consisting of N,N-diisopropylethylamine (DIPEA), triethylamine (TEA), potassium tert-butoxide (t-BuOK) and pyridine (Py), the inorganic base can be selected from potassium phosphate (K ₃ PO4), sodium hydride (NaH), potassium carbonate (K ₂ CO ₃ ), carbonic acid Sodium (Na ₂ CO ₃ ), sodium bicarbonate (NaHCO ₃ ), cesium carbonate (Cs ₂ CO ₃ ) and NaOH, preferably Na ₂ CO ₃ or NaHCO ₃ ;

Step 7

The compound of formula (IV) is obtained by the condensation reaction of the compound of formula (IV)-IM16 and the compound of formula (IV)-SM4;

In some embodiments, this step is performed under a suitable condensation reagent, which can be selected from HATU, HBTU, EDCI, DCC and HOBT, preferably HBTU;

In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 25°C;

In some embodiments, this step is performed in a suitable organic solvent, which may be selected from the group consisting of methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl Sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably N,N-dimethylformamide.

In some embodiments, this step is carried out in a suitable base, including an organic base or an inorganic base, the organic base can be selected from DIPEA, TEA, t-BuOK and Py, and the inorganic base can be selected from K ₃ PO ₄ , NaH, K ₂ CO ₃ , Na ₂ CO ₃ , Cs ₂ CO ₃ and NaOH, preferably DIPEA.

The compound of formula (V)-1 in the present invention can use starting materials Synthetic preparation is carried out according to the same synthetic route as formula (III).

Beneficial effects of the invention

The present invention provides camptothecin compounds represented by formula (I) to formula (IV), pharmaceutical compositions, preparation methods and uses thereof. This type of compound has good anti-tumor activity, has the potential to overcome drug resistance, and can be used to treat abnormal cell proliferation disorders, including but not limited to advanced solid tumors.

Detailed ways

The present application will be further described below through the description of specific embodiments, but this is not intended to limit the present application. Those skilled in the art can make various modifications or improvements based on the teachings of this application without departing from the basic idea and scope of this application.

The abbreviations in this invention have the following meanings:

The structures of the compounds described in the following examples were determined by nuclear magnetic resonance ( ¹ H NMR) or mass spectrometry (MS).

The measuring instrument of nuclear magnetic resonance ( ¹ H NMR) used a Bruker 400 MHz nuclear magnetic resonance instrument; hexadeuterated dimethyl sulfoxide (DMSO-d ₆ ); the internal standard substance was tetramethylsilane (TMS).

The abbreviations in the nuclear magnetic resonance (NMR) spectra used in the examples are shown below.

s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br: broad, J: coupling constant, Hz: Hertz, DMSO-d6: deuterated dimethyl sulfoxide. The delta value is expressed as ppm value.

The mass spectrometry (MS) measurement instrument used was an Agilent (ESI) mass spectrometer, model Agilent 6120B.

Example 1 (R)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10, 13,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2 -Hydroxypent-3-ynamide and (S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2, 3,9,10,13,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline- 1-yl)-2-hydroxypent-3-ynamide

Compound 2-hydroxypent-3-ynoic acid (4.29 mg, 37.63 μmol) was dissolved in DMF (1 mL), and HATU (21.46 mg, 56.44 μmol), SM1-1 (10.00 mg, 22.94 μmol) and DIPEA (7.29 mg were added ,56.44μmol), react at 25℃ for 2 hours. The reaction solution was concentrated under reduced pressure, and the concentrate was directly purified by preparative high-performance liquid chromatography (conditions are as follows) to obtain 3.24 mg of the title compounds 1-1-A and 3.98 mg of 1-1-B.

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

time[min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL/min] 0 30 70 28 18 90 10 28

Retention time: 1-1-A: 10.8min; 1-1-B: 11.1min.

The structural characterization data of 1-1-A are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.58(d,J＝8.4Hz,1H),7.78(d,J＝11.2Hz,1H),7.31(s,1H),6.54(s,1H) ,6.17(d,J＝6.0Hz,1H),5.58-5.47(m,1H),5.42(s,2H),5.23(s,2H),4.73-4.64(m,1H),3.25-3.06(m ,2H),2.39(s,3H),2.27-2.05(m,2H),1.96-1.77(m,5H),0.87(t,J＝7.2Hz,3H).

ESI-MS(m/z):532.2[M+H] ⁺ .

The structural characterization data of 1-1-B are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.62 (d, J = 8.8 Hz, 1H), 7.78 (d, J = 10.8 Hz, 1H), 7.31 (s, 1H), 6.54 (s, 1H) ,6.19(d,J＝6.0Hz,1H),5.58-5.47(m,1H),5.42(s,2H),5.21(d,J＝4.8Hz,2H),4.73-4.65(m,1H), 3.27-3.06(m,2H),2.39(s,3H),2.27-2.05(m,2H),1.93-1.80(m,2H),1.80(d,J＝2.0Hz,3H),0.87(t, J＝7.2Hz,3H).

ESI-MS(m/z):532.2[M+H] ⁺ .

Example 2 (S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10, 13,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4]:6,7]indolizino[1,2-b]quinolin-1-yl)-2 -Hydroxy-3-enamide and (R)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3 ,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4]:6,7]indolizino[1,2-b]quinoline-1 -yl)-2-hydroxy-3-enamide

The compound ethylene glycolic acid (9.61 mg, 94.07 μmol) was dissolved in DMF (2 mL), and HATU (44.70 mg, 117.58 μmol), compound SM1-1 (25.00 mg, 0.047 mmol) and DIPEA (24.30 mg, 188.13 μmol) were added. React at 25°C for 2 hours. The reaction solution was concentrated under reduced pressure, and the concentrate was directly purified by preparative high-performance liquid chromatography to obtain the title compounds 1-7-A (4.00 mg) and 1-7-B (1.38 mg).

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

time[min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL/min] 0 30 70 28

2 30 70 28 18 90 10 28

Retention time: 1-7-A: 8.7min; 1-7-B: 9.1min.

The structural characterization data of 1-7-A are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.51 (s, J = 8.8 Hz, 1H), 7.76 (s, J = 10.8 Hz, 1H), 7.29 (s, 1H), 6.52 (s, 1H) ,6.15–6.04(m,1H),5.58–5.49(m,1H),5.42(s,2H),5.39(s,1H),5.24–5.01(m,3H),4.54(s,J＝4.9Hz ,1H),3.24–3.05(m,2H),2.37(s,3H),2.16(s,2H),1.91–1.79(m,2H),0.87(t,J＝7.2Hz,3H).

ESI-MS(m/z):520.1[M+H] ⁺ .

The structural characterization data of 1-7-B are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.48(s,1H),7.78(s,1H),7.30(s,1H),6.12–5.92(m,1H),5.54–5.47(m,1H) ),5.42(s,2H),5.37(dt,J＝1.7Hz,1H),5.18(s,2H),5.16–5.14(m,1H),4.5–4.52(m,1H),3.22–3.08( m,2H),2.38(s,3H),2.25–2.16(m,1H),2.16–2.06(m,1H),1.93–1.79(m,2H),0.87(t,J＝7.2Hz,3H) .

ESI-MS(m/z):520.1[M+H] ⁺ .

Example 3 N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15- Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide and N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro -1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide

Step 1: Synthesis of 1-chloro-3-bromo-2-methyl-5-nitrobenzene

Dissolve compound 2-1-01 (5.00g, 29.14mmol) in n-heptane (25mL) at 25°C, add concentrated sulfuric acid (25mL), heat to 50°C, add NBS (6.22) in batches at 50°C g, 34.97 mmol), keep the reaction at 50°C for 2 hours, use thin layer chromatography to detect the reaction (ethyl acetate: petroleum ether = 1:10), add the reaction liquid cooled to room temperature dropwise into ice water, extract with toluene, and combine the organic phases , washed with sodium sulfite solution, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, the crude product was purified by preparative high performance liquid chromatography, and the prepared solution was freeze-dried to obtain 4.88g of the title compound.

Column: C18 ODS 45mm×450mm×8.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

time[min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL/min] 0 60 40 60 10 60 40 60 40 100 0 60

Step 2: Synthesis of 3-chloro-5-bromo-4-methylaniline

Dissolve compound 2-1-02 (4.88g, 19.48mmol) in ethyl acetate (100mL) at 25°C, add platinum carbon (2.00g, 19.48mmol, content 5%), replace it with hydrogen and protect it with a hydrogen balloon. The reaction was carried out at 60°C for 4 hours, and the reaction was monitored by high-performance liquid chromatography-mass spectrometry. The reaction solution was filtered, and the filtrate was concentrated to obtain 3.68 g of crude title compound, which was directly used in the next reaction without further purification.

Step 3: Synthesis of N-(3-chloro-5-bromo-4-methylphenyl)acetamide

Dissolve compound 2-1-03 (3.63g, 14.82mmol) in ethyl acetate (70mL) at 20°C, add triethylamine (4.50g, 44.45mmol) and acetic anhydride (2.27g, 22.23mmol), and keep The reaction was carried out at 20°C for 20 hours, and the reaction was monitored by high-performance liquid chromatography-mass spectrometry. Water was added to the reaction solution and extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product. The crude product was slurried with a mixed solvent of ethyl acetate:petroleum ether = 1:5 to obtain 2.86 g of the title compound.

Step 4: Synthesis of (Z)-4-(5-acetamido-3-chloro-2-methylphenyl)but-3-enoic acid

Dissolve compound 2-1-04 (1.80g, 6.86mmol) in THF (20mL) and water (5mL) at 20°C, add vinyl acetic acid (708.31mg, 8.23mmol), DIPEA (1.95g, 15.08mmol) ), tris(o-methylphenyl)phosphorus (62.60 mg, 0.20 mmol), the reaction system was replaced with nitrogen and heated to 70°C for 5 hours, and the reaction was monitored by high-performance liquid chromatography-mass spectrometry. 1N sodium hydroxide solution was added to the reaction solution to adjust pH=8, and ethyl acetate was added for extraction. The remaining aqueous phase was adjusted to pH=3 with 1N hydrochloric acid, extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 0.82g of the title compound, which was directly used in the next reaction.

Step 5: Synthesis of 4-(5-acetamido-3-chloro-2-methylphenyl)butyric acid

Dissolve compound 2-1-05 (2.60g, 9.71mmol) in THF (50mL) at 20°C, add Pd/C (0.52g, content 10%), and replace the system with hydrogen for 40 seconds under the protection of a hydrogen balloon. The reaction was carried out for 2 hours at ℃, and the reaction was monitored by high performance liquid chromatography-mass spectrometry. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain 2.43 g of the title compound, which was directly used in the next reaction without further purification.

Step 6: Synthesis of N-(3-chloro-4-methyl-8-oxo-5,6,7,8-tetralin-1-yl)acetamide

Dissolve compound 2-1-06 (2.43g, 9.01mmol) in trifluoroacetic acid (10mL), cool to 5°C, add trifluoroacetic anhydride (3.78g, 18.02mmol, 2.50mL) dropwise, and maintain the reaction at 5°C. After 4 hours, the reaction was monitored using HPLC-MS chromatography. Add the reaction solution to water, dissolve it in 10N sodium hydroxide to adjust pH = 9, add ethyl acetate for extraction, combine the organic phases, dry over anhydrous sodium sulfate, concentrate under reduced pressure, and purify through a fast silica gel column (ethyl acetate:petroleum) Ether = 0-20%) to obtain 1.53 g of the title compound.

Step 7: (Z)-N-(3-chloro-7-(hydroxyimino)-4-methyl-8-oxo-5,6,7,8-tetralin-1-yl)acetamide Synthesis of Potassium tert-butoxide (1.50g, 13.37mmol) was dissolved in THF (16mL) and tert-butanol (4mL) at 5°C, and the THF of compound 2-1-07 (1.53g, 6.08mmol) was added dropwise. solution (16 mL), add amyl nitrite (1.14 g, 9.73 mmol) dropwise after 10 minutes, keep the reaction at 5°C for 1 hour, and monitor the reaction with high-performance liquid chromatography-mass spectrometry. The reaction solution was adjusted to pH=5 with 1N hydrochloric acid and extracted with ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The concentrate was slurried with methyl tert-butyl ether to obtain 1.20 g of the title compound.

Step 8: N-(7-amino-3-chloro-4-methyl-8-oxo-5,6,7,8-tetralin-1-yl)acetamide

At 20°C, compound 2-1-08 (0.50g, 1.78mmol) was dissolved in methanol (8mL) and 2N hydrochloric acid (8mL), Pd/C (0.15g, content 10%) was added, and the system was replaced with hydrogen. Keep the reaction at 5°C for 2 hours under the protection of a hydrogen balloon, and monitor the reaction with high-performance liquid chromatography-mass spectrometry. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain 0.52 g of the hydrochloride of the title compound, which was directly used in the next reaction without further purification.

Step 9: Synthesis of N,N’-(3-chloro-4-methyl-8-oxo-5,6,7,8-tetralin-1,7-diyl)diethylamide

Dissolve compound 2-1-09 (0.52g, 1.70mmol) in pyridine (5mL) at 20°C, add acetic anhydride (2mL), keep the reaction at 20°C for 2 hours, and monitor the reaction with high-performance liquid chromatography-mass spectrometry. . The reaction solution was added to water, extracted with ethyl acetate, the organic phases were washed with water, combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the concentrate was purified by a fast silica gel column (ethyl acetate: petroleum ether = 0-30%) to obtain Title compound 0.22g.

Step 10: Synthesis of N-(8-amino-6-chloro-5-methyl-1-oxo-1,2,3,4-tetralin-2-yl)acetamide

Dissolve compound 2-1-10 (0.45g, 1.46mmol) in methanol (16mL) at 20°C, add 2N hydrochloric acid (16mL), heat to 60°C, react for 2 hours, and monitor with high-performance liquid chromatography-mass spectrometry. reaction. Add saturated sodium bicarbonate solution to the cooled reaction solution to adjust pH=8, extract with ethyl acetate, combine the organic phases, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain 0.23g of the title compound, which was used directly without further purification. react in the next step.

Step 11: N-((9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-six Synthesis of hydrogen-1H,12H-benzopyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide

Compound 2-1-11 (0.23g, 0.78mmol) was dissolved in toluene (10mL), and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3 ,4-f]indolazine-3,6,10(4H)-trione (0.23g, 0.87mmol), p-toluenesulfonic acid (26.73mg, 0.16mmol), heated to 140°C for 5 hours, The reaction was monitored using HPLC-MS chromatography. The reaction solution was concentrated, and the crude product was purified by flash silica gel column (methanol:dichloromethane=0-10%) to obtain 0.15g of the title compound.

Step 12: (9S)-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzene Synthesis of pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione

Compound 2-1-12 (40.00 mg, 0.08 mmol) was added to concentrated hydrochloric acid (1 mL), heated to 100°C for 5 hours, and the reaction was monitored using high-performance liquid chromatography-mass spectrometry. The reaction solution was filtered, and the filtrate was purified by preparative high-performance liquid chromatography. The prepared solution was freeze-dried to obtain 12.00 mg of the trifluoroacetate salt of the title compound 2-23.

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% trifluoroacetic acid)

time[min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL/min] 0 5 95 28 2 5 95 28 18 50 50 28

The structural characterization data are as follows:

ESI-MS(m/z):452.1[M+H] ⁺ .

Step 13: 2-((tert-butyldiphenylsilyl)oxy)-N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-1-yl)acetamide and 2-((tert-butyldiphenylsilyl)oxy)-N-((1R,9S)-5-chloro-9-ethyl- 9-Hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4' :Synthesis of 6,7]indolizino[1,2-b]quinolin-1-yl)acetamide

At 25°C, the trifluoroacetate salt of compound 2-23 (40.00 mg, 81.91 μmol) was dissolved in N,N-dimethylformamide (1 mL), and 2-((tert-butyldiphenyl) was added in sequence. Silyl)oxy)acetic acid (30.91mg, 98.29μmol), HATU (62.25mg, 163.81μmol) and N,N-diisopropylethylamine (42.34mg, 327.63μmol) were kept at 25°C for 0.5 hours and reacted with High-performance liquid chromatography-mass spectrometry coupled to chromatography monitored the reaction. After the reaction is completed, water is added to the reaction solution, extracted with dichloromethane/methanol (v/v=10/1), the organic phases are combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the crude product is purified by preparative thin layer chromatography ( Dichloromethane: methanol = 20:1), two isomers were separated, and the two isomers were named 2-1-13-A (15.00 mg, Rf value 0.3) and 2-1- based on the Rf value. 13-B (12.00mg, Rf value 0.35).

Step 14: N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15 -Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyethyl Amide and N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexa Hydrogen-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide synthesis

Dissolve 2-1-13-A (15.00mg) and 2-1-13-B (12.00mg) in tetrahydrofuran (1mL) in two reaction bottles at 25°C, and add tetrabutyl fluoride dropwise. Ammonium (1M tetrahydrofuran solution)/glacial acetic acid mixture (v/v=13/1) (50uL), keep the reaction at 25°C for 0.5 hours, and monitor the reaction with high-performance liquid chromatography-mass spectrometry. After the reaction was completed, the reaction solution was purified by preparative high-performance liquid chromatography, and the preparation solution was freeze-dried to obtain the title compounds 2-1-A (6.94 mg) and 2-1-B (4.00 mg).

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

time[min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL/min] 0 20 80 28 3 20 80 28 18 90 10 28

The structural characterization data of 2-1-A are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.43 (d, J = 8.8Hz, 1H), 8.16 (s, 1H), 7.31 (s, 1H), 6.55 (s, 1H), 5.65-5.36 ( m,4H),5.21(q,J＝19.0Hz,2H),3.95(d,J＝5.7Hz,2H),3.26-3.11(m,2H),2.53(s,3H),2.30-2.08(m ,2H),1.94-1.79(m,2H),0.87(t,J＝7.3Hz,3H).

ESI-MS(m/z):510.1[M+H] ⁺ .

The structural characterization data of 2-1-B are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.45 (d, J = 8.9 Hz, 1H), 8.15 (s, 1H), 7.31 (s, 1H), 6.54 (s, 1H), 5.64-5.35 ( m,4H),5.19(q,J＝19.0Hz,2H),3.97(d,J＝5.2Hz,2H),3.27-3.10(m,2H),2.51(s,3H),2.27-2.10(m ,2H),1.93-1.80(m,2H),0.88(t,J=7.3Hz,3H).

ESI-MS(m/z):510.1[M+H] ⁺ .

Example 4 (2S)-N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10, 13,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2 -Hydroxypropylamine and (2S)-N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10 ,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)- 2-Hydroxypropylamine

Step 1: (2S)-2-((tert-butyldiphenylsilyl)oxy)-N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl -10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazine And[1,2-b]quinolin-1-yl)propylamine and (2S)-2-((tert-butyldiphenylsilyl)oxy)-N-((1R,9S)-5-chloro -9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano Synthesis of [3',4':6,7]indolizino[1,2-b]quinolin-1-yl)propylamine

Dissolve the hydrochloride of 2-23 (30.00 mg, 61.43 μmol) in N,N-dimethylformamide (1 mL) at 25°C, and add (S)-2-((tert-butyldiphenyl) in sequence Si)oxy)propionic acid (24.21mg, 73.72μmol), HATU (35.01mg, 92.14μmol) and N,N-diisopropylethylamine (23.82mg, 184.29μmol), kept at 25°C for 1 hour, The reaction was monitored using HPLC-MS chromatography. After the reaction is completed, water is added to the reaction solution, extracted with dichloromethane/methanol (v/v=10/1), the organic phases are combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the crude product is purified by preparative thin layer chromatography ( Dichloromethane: methanol = 15:1) were separated to obtain two isomers, which were named 2-7-01-A (6.00 mg, Rf value 0.35) and 2-7- based on the Rf value. 01-B (6.00mg, Rf value 0.40).

Step 2: (2S)-N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10, 13,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2 -Hydroxypropylamine and (2S)-N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10 ,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)- Synthesis of 2-Hydroxypropylamine

Dissolve 2-7-01-A (6.00 mg, 7.87 μmol) and 2-7-01-B (6.00 mg, 7.87 μmol) in anhydrous tetrahydrofuran (1 mL) in two reaction bottles at 25°C. , add dropwise tetrabutylammonium fluoride (1M tetrahydrofuran solution)/glacial acetic acid mixture (v/v=13/1) (50 μL), keep the reaction at 25°C for 0.5 hours, and monitor the reaction with high-performance liquid chromatography-mass spectrometry. After the reaction was completed, the reaction solution was purified by preparative high performance liquid chromatography, and the preparation solution was freeze-dried to obtain the title compounds 2-7-A (2.50 mg) and 2-7-B (3.00 mg).

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

time[min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL/min] 0 15 85 28 16 90 10 28

The structural characterization data of 2-7-A are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.38 (d, J = 8.8 Hz, 1H), 8.15 (s, 1H), 7.31 (s, 1H), 6.55 (s, 1H), 5.56-5.47 ( m,2H),5.42(s,2H),5.26-5.11(m,2H),4.17-4.06(m,1H),3.27-3.10(m,2H),2.52(s,3H),2.27-2.08( m,2H),1.86(tt,J＝14.1,7.3Hz,2H),1.30(d,J＝6.7Hz,3H),0.87(t,J＝7.3Hz,3H).

ESI-MS(m/z):524.2[M+H] ⁺ .

The structural characterization data of 2-7-B are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.48 (d, J = 9.1 Hz, 1H), 8.12 (s, 1H), 7.30 (s, 1H), 6.54 (s, 1H), 5.67 (d, J＝4.8Hz,1H),5.55(dd,J＝14.6,7.3Hz,1H),5.43(s,2H),5.24(d,J＝19.0Hz,1H),5.03(d,J＝19.0Hz, 1H),4.21-4.08(m,1H),3.28-3.08(m,2H),2.51(s,3H),2.16(d,J＝6.2Hz,2H),1.94-1.82(m,2H),1.42 (d,J＝6.8Hz,3H),0.88(t,J＝7.3Hz,3H).

ESI-MS(m/z):524.2[M+H] ⁺ .

Example 5 (2S)-N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10, 13,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2 -Cyclopropyl-2-hydroxyacetamide and (2S)-N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quin Phin-1-yl)-2-cyclopropyl-2-hydroxyacetamide and (2R)-N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl- 10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino [1,2-b]quinolin-1-yl)-2-cyclopropyl-2-hydroxyacetamide and (2R)-N-((1R,9S)-5-chloro-9-ethyl-9 -Hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4': 6,7]indolizino[1,2-b]quinolin-1-yl)-2-cyclopropyl-2-hydroxyacetamide

Step 1: (2S)-2-((tert-butyldiphenylsilyl)oxy)-N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl- 10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino [1,2-b]quinolin-1-yl)-2-cyclopropylacetamide and (2S)-2-((tert-butyldiphenylsilyl)oxy)-N-((1R,9S )-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[ de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-cyclopropylacetamide and (2R)-2-( (tert-butyldiphenylsilyl)oxy)-N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2, 3,9,10,13,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline- 1-yl)-2-cyclopropylacetamide and (2R)-2-((tert-butyldiphenylsilyl)oxy)-N-((1R,9S)-5-chloro-9-ethyl -9-Hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4 Synthesis of ':6,7]indolizino[1,2-b]quinolin-1-yl)-2-cyclopropylacetamide

Dissolve the hydrochloride of 2-23 (30.00 mg, 61.43 μmol) in N,N-dimethylformamide (1 mL) at 25°C, and add 2-((tert-butyldiphenylsilyl)) Oxy)-2-cyclopropylacetic acid (26.13mg, 73.72μmol), HATU (35.01mg, 92.14μmol) and N,N-diisopropylethylamine (23.82mg, 184.29μmol), keep at 25°C for reaction 1 hours, and the reaction was monitored using high-performance liquid chromatography-mass spectrometry. After the reaction is completed, water is added to the reaction solution, extracted with dichloromethane/methanol (v/v=10/1), the organic phases are combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the crude product is purified by preparative thin layer chromatography ( Dichloromethane: methanol = 15:1), two sets of isomers were separated and named 2-12-01-A (8.00 mg, Rf value 0.35) and 2-12- based on the Rf value. 01-B (10.00mg, Rf value is 0.40).

Step 2: (2S)-N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10, 13,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2 -Cyclopropyl-2-hydroxyacetamide and (2S)-N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quin Phin-1-yl)-2-cyclopropyl-2-hydroxyacetamide and (2R)-N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl- 10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino [1,2-b]quinolin-1-yl)-2-cyclopropyl-2-hydroxyacetamide and (2R)-N-((1R,9S)-5-chloro-9-ethyl-9 -Hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4': Synthesis of 6,7]indolizino[1,2-b]quinolin-1-yl)-2-cyclopropyl-2-hydroxyacetamide

Dissolve 2-12-01-A (8.00 mg, 10.15 μmol) and 2-12-01-B (10.00 mg, 12.68 μmol) in anhydrous tetrahydrofuran (1 mL) in two reaction bottles at 25°C. , add dropwise tetrabutylammonium fluoride (1M tetrahydrofuran solution)/glacial acetic acid mixture (v/v=13/1) (50 μL), keep the reaction at 25°C for 0.5 hours, and monitor the reaction with high-performance liquid chromatography-mass spectrometry. After the reaction is completed, the reaction solution is purified by preparative high-performance liquid chromatography. The reaction using 2-12-01-A as the raw material separates two isomer products. The preparation solution is freeze-dried to obtain compound 2-12-A (0.77 mg), 2-12-B (1.03mg); 2-12-01-B was used as the raw material to separate two isomer products, and the preparation solutions were freeze-dried to obtain compound 2-12-C (2.50mg). , 2-12-D (1.00mg). 2-12-A/2-12-B purification conditions are as follows:

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

time[min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL/min] 0 20 80 28

2 20 80 28 18 80 20 28

Peak retention time is: 2-12-A: 10.0-11.0min, 2-12-B: 11.0-12.5min

2-12-C/2-12-D purification conditions are as follows:

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

time[min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL/min] 0 20 80 28 2 20 80 28 18 80 20 28

The peak retention time is: 2-12-C: 10.6-11.4min, 2-12-D: 11.4-12.5min

The structural characterization data of 2-12-A are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.38 (d, J = 8.9 Hz, 1H), 8.15 (s, 1H), 7.30 (s, 1H), 6.54 (s, 1H), 5.63-5.53 ( m,1H),5.51(d,J＝5.1Hz,1H),5.42(s,2H),5.28(d,J＝19.2Hz,1H),5.16(d,J＝19.1Hz,1H),3.60( t,J＝5.6Hz,1H),3.28-3.11(m,2H),2.52(s,3H),2.22-2.10(m,2H),1.86(tt,J＝14.1,7.3Hz,2H),1.23 (d,J＝4.9Hz,1H),0.87(t,J＝7.2Hz,3H),0.56-0.36(m,4H).

ESI-MS(m/z):550.2[M+H] ⁺ .

The structural characterization data of 2-12-B are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.36 (d, J = 8.7Hz, 1H), 8.16 (s, 1H), 7.31 (s, 1H), 6.55 (s, 1H), 5.57-5.48 ( m,1H),5.42(s,2H),5.40(d,J＝5.4Hz,1H),5.26(d,J＝19.3Hz,1H),5.18(d,J＝19.0Hz,1H),3.65- 3.60(m,1H),3.26-3.12(m,2H),2.52(s,3H),2.26-2.09(m,2H),1.86(tt,J＝14.1,7.2Hz,2H),1.19-1.08( m,1H),0.87(t,J=7.3Hz,3H),0.51-0.27(m,4H).

ESI-MS(m/z):550.2[M+H] ⁺ .

The structural characterization data of 2-12-C are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.42 (d, J = 9.0 Hz, 1H), 8.15 (s, 1H), 7.31 (s, 1H), 6.54 (s, 1H), 5.56 (dd, J＝14.8,6.8Hz,1H),5.52(d,J＝5.2Hz,1H),5.42(s,2H),5.29(d,J＝19.2Hz,1H),5.16(d,J＝19.1Hz, 1H),3.62-3.58(m,1H),3.27-3.08(m,2H),2.51(s,3H),2.27-2.08(m,2H),1.87(tt,J＝14.0,7.2Hz,2H) ,1.25(dd,J＝13.2,6.8Hz,1H),0.87(t,J＝7.3Hz,3H),0.64-0.28(m,4H).

ESI-MS(m/z):550.1[M+H] ⁺ .

The 2-12-D structural characterization data are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.38 (d, J = 8.7Hz, 1H), 8.16 (s, 1H), 7.31 (s, 1H), 6.54 (s, 1H), 5.59-5.49 ( m,1H),5.43(d,J＝5.3Hz,1H),5.43(s,2H),5.26(d,J＝19.1Hz,1H), 5.17(d,J＝19.0Hz,1H),3.64( t,J＝5.8Hz,1H),3.17(dd,J＝15.6,8.3Hz,2H),2.27-2.07(m,2H),1.94-1.79(m,2H),1.19-1.06(m,1H) ,0.87(t,J=7.3Hz,3H),0.49-0.28(m,4H).

ESI-MS(m/z):550.1[M+H] ⁺ .

Example 6 N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15- Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxy-2 -Methylpropylamine and N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13, 15-Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxy -2-Methylpropylamine

Dissolve the hydrochloride of 2-23 (30.00 mg, 61.43 μmol) in N,N-dimethylformamide (1 mL) at 25°C, and add 2-((tert-butyldimethylsilyl)) in sequence Oxy)-2-methylpropionic acid (16.10mg, 73.72μmol), HATU (35.01mg, 92.14μmol) and N,N-diisopropylethylamine (23.82mg, 184.29μmol), keep at 25°C for reaction 1 hours, and the reaction was monitored using high-performance liquid chromatography-mass spectrometry. After the reaction was completed, the reaction solution was concentrated, and the crude product was purified and separated by preparative high-performance liquid chromatography to obtain two isomers. The preparation solutions were freeze-dried respectively. The two isomers were named 2-17-A (2.65) based on the peak retention time. mg) and 2-17-B (2.69 mg).

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

time[min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL/min] 0 20 80 28 2 20 80 28 18 80 20 28

The peak retention times are: 2-17-A: 9.5-10.2min and 2-17-B: 10.4-10.6min.

The structural characterization data of 2-17-A are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.36 (d, J = 9.1Hz, 1H), 8.13 (s, 1H), 7.30 (s, 1H), 6.54 (s, 1H), 5.55-5.45 ( m,2H),5.42(s,2H),5.28(d,J＝19.0Hz,1H),5.05(d,J＝19.0Hz,1H),3.27-3.11(m,2H),2.51(s,1H ),2.24-2.10(m,2H),1.86(tt,J＝14.0,7.2Hz,2H),1.46(s,3H),1.35(s,3H),0.87(t,J＝7.3Hz,3H) .

MS m/z(ESI):538.2[M+H] ⁺ .

The structural characterization data of 2-17-B are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.40 (d, J = 9.2Hz, 1H), 8.13 (s, 1H), 7.30 (s, 1H), 6.53 (s, 1H), 5.60-5.46 ( m,2H),5.42(s,2H),5.29(d,J＝19.0Hz,1H),5.02(d,J＝19.0Hz,1H),3.28-3.08(m,2H),2.50(s,3H ),2.22-2.10(m,2H),1.87(tt,J＝14.2,7.2Hz,2H),1.47(s,3H),1.35(s,3H),0.88(t,J＝7.3Hz,3H) .

MS m/z(ESI):538.2[M+H] ⁺ .

Example 7 N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15- Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-hydroxycyclopropane -1-Carboxamide and N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13 ,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1- Hydroxycyclopropane-1-carboxamide

Step 1: 1-((tert-butyldiphenylsilyl)oxy)-N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13 -Dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1, 2-b]quinolin-1-yl)cyclopropane-1-carboxamide and 1-((tert-butyldiphenylsilyl)oxy)-N-((1R,9S)-5-chloro-9 -Ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3 Synthesis of ',4':6,7]indolizino[1,2-b]quinolin-1-yl)cyclopropane-1-carboxamide

Dissolve the hydrochloride of 2-23 (30.00mg, 61.43umol) in N,N-dimethylformamide (1mL) at 25°C, and add 1-((tert-butyldiphenylsilyl)) Oxy)cyclopropane-1-carboxylic acid (25.10mg, 73.72umol), HATU (35.01mg, 92.14umol) and N,N-diisopropylethylamine (23.82mg, 184.29μmol), keep at 25°C for reaction 1 hours, and the reaction was monitored using high-performance liquid chromatography-mass spectrometry. After the reaction is completed, water is added to the reaction solution, extracted with dichloromethane/methanol (v/v=10/1), the organic phases are combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the crude product is purified by preparative thin layer chromatography ( Dichloromethane: methanol = 15:1) were separated to obtain two isomers, which were named 2-20-01-A (4.00 mg, Rf value 0.30) and 2-20- based on the Rf value. 01-B (4.00mg, Rf value is 0.35).

Step 2: N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15- Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-hydroxycyclopropane -1-Carboxamide and N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13 ,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1- Synthesis of Hydroxycyclopropane-1-carboxamide

Dissolve 2-20-01-A (4.00 mg, 5.17 μmol) and 2-20-01-B (4.00 mg, 5.17 μmol) in anhydrous tetrahydrofuran (1 mL) in two reaction bottles at 25°C. , add dropwise tetrabutylammonium fluoride (1M tetrahydrofuran solution)/glacial acetic acid mixture (v/v=13/1) (50 μL), keep the reaction at 25°C for 0.5 hours, and monitor the reaction with high-performance liquid chromatography-mass spectrometry. After the reaction was completed, the reaction solution was purified by preparative high-performance liquid chromatography, and the preparation solution was freeze-dried to obtain the title compounds 2-20-A (0.71 mg) and 2-20-B (1.05 mg).

2-20-A purification conditions are as follows:

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

time[min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL/min] 0 15 85 28 16 90 10 28

2-20-B purification conditions are as follows:

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

time[min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL/min] 0 20 80 28 2 20 20 28 18 80 20 28

The structural characterization data of 2-20-A are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.58 (d, J = 9.0 Hz, 1H), 8.15 (s, 1H), 7.31 (s, 1H), 6.55 (s, 1H), 6.30 (s, 1H),5.55(dd,J＝13.2,8.2Hz,1H),5.43(s,2H),5.26(d,J＝19.0Hz,1H),5.10(d,J＝19.0Hz,1H),3.29- 3.09(m,2H),2.52(s,3H),2.31-2.15(m,2H),1.93-1.80(m,2H),1.25-1.14(m,2H),0.98-0.90(m,2H), 0.87(t,J＝7.3Hz,3H).

ESI-MS(m/z):536.2[M+H] ⁺ .

The structural characterization data of 2-20-B are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.63 (d, J = 9.0Hz, 1H), 8.15 (s, 1H), 7.31 (s, 1H), 6.54 (s, 1H), 6.35 (s, 1H),5.55(dd,J＝13.5,8.6Hz,1H),5.43(s,2H),5.29(d,J＝19.1Hz,1H),5.08(d,J＝19.1Hz,1H),3.28- 3.10(m,2H),2.51(s,3H),2.30-2.14(m,2H),1.93-1.81(m,2H),1.26-1.14(m,2H),1.02-0.90(m,2H), 0.89(d,J=10.9Hz,3H).

ESI-MS(m/z):536.2[M+H] ⁺ .

Example 8 (1S,9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzene And[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione and (1R,9S)-1-amino-4 -Chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4': 6,7]indolizino[1,2-b]quinoline-10,13-dione

Step 1: Synthesis of 3-bromo-4-chloro-5-fluoroaniline

Compound 3-1-01 (2.00g, 10.53mmol) was dissolved in N,N-dimethylformamide (30mL), and then N-chlorosuccinimide (1.69g, 12.63mmol) was slowly added. After the addition was completed, the reaction was carried out at room temperature for 16 hours, and the reaction was detected by high performance liquid chromatography mass spectrometry coupled with chromatography. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was purified by a fast silica gel column (ethyl acetate: petroleum ether = 0-25%) to obtain 0.95g of the title compound.

The structural characterization data are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ6.77 (dd, J = 2.5, 1.4 Hz, 1H), 6.51 (dd, J = 11.7, 2.5

Hz,1H),5.84(s,2H).

Step 2: Synthesis of N-(3-bromo-4-chloro-5-fluorophenyl)acetamide

Compound 3-1-02 (0.95g, 4.23mmol) was dissolved in ethyl acetate (20mL), and acetic anhydride (648.13mg, 6.35mmol) was added under nitrogen protection. After the addition was completed, the temperature was raised to 50°C and the reaction was carried out for 15 hours. High-performance liquid chromatography-mass spectrometry coupled to chromatography detects the reaction. After the reaction solution was quenched with methanol (5 mL), it was directly evaporated to dryness under reduced pressure to obtain a crude product. The crude product was purified by a flash silica gel column (ethyl acetate: petroleum ether = 0-40%) to obtain 1.01 g of the title compound.

The structural characterization data are as follows:

ESI-MS(m/z):265.9[M+H] ⁺ .

Step 3: Synthesis of (E)-4-(5-acetylamino-2-chloro-3-fluorophenyl)-3-butenoic acid

Compound 3-1-03 and 3-butenoic acid (387.65 mg, 4.50 mmol) were dissolved in a mixed solvent of 1,4 dioxane (24 mL) and water (8 mL), and then N, N-diiso After adding propylethylamine (1.45g, 11.26mmol), tris(o-methylphenyl)phosphorus (114.21mg, 375.24μmol) and palladium acetate (42.12mg, 187.62μmol), the reaction system was replaced with nitrogen three times, and The temperature was raised to 100°C and reacted for 16 hours under a nitrogen atmosphere, and the reaction was detected by high-performance liquid chromatography-mass spectrometry. After the reaction solution was cooled to room temperature, 1N aqueous sodium hydroxide solution (60 mL) and ethyl acetate (50 mL) were added and the mixture was separated by shaking. After separating the lower aqueous phase, adjust the pH to about 3 with 4 mol/L hydrochloric acid aqueous solution, and then extract with ethyl acetate. The combined organic phases are washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate is evaporated to dryness under reduced pressure. 1.00 g of crude product of the title compound was obtained.

The structural characterization data are as follows:

ESI-MS(m/z):272.0[M+H] ⁺ .

Step 4: Synthesis of 4-(5-acetamido-2-chloro-3-fluorophenyl)butyric acid

Dissolve the crude product of compound 3-1-04 (1.00g, 3.68mmol) in tetrahydrofuran (15mL), then add 10% palladium on carbon (0.10g), complete the addition, and then replace the reaction system with a hydrogen balloon three times, and in a hydrogen atmosphere The reaction was carried out for 4 hours, and the reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain 1.00 g of crude product of the title compound.

The structural characterization data are as follows:

ESI-MS(m/z):274.0[M+H] ⁺ .

Step 5: Synthesis of N-(4-chloro-3-fluoro-8-oxo-5,6,7,8-tetralin-1-yl)acetamide

The crude product of compound 3-1-05 (1.00g, 3.65mmol) was dissolved in trifluoroacetic acid (5mL). After cooling to 5°C, trifluoroacetic anhydride (3.84g, 18.27mmol, 2.54mL) was slowly added. After completion, the reaction was maintained at 5°C for 2 hours, and the reaction was detected by high-performance liquid chromatography-mass spectrometry. The reaction solution was slowly poured into water, and then extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered. The filtrate was evaporated to dryness under reduced pressure to obtain a crude product, which was purified by a fast silica gel column. , 0.43g of the title compound was obtained.

The structural characterization data are as follows:

ESI-MS(m/z):256.1[M+H] ⁺ .

Step 6: Synthesis of N-(4-chloro-3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetralin-1-yl)acetamide

Add tetrahydrofuran (16 mL) and tert-butyl alcohol (4 mL) to the reaction solution bottle, cool it to 5°C in an ice bath, add potassium tert-butoxide (415.18 mg, 3.70 mmol), and then add compound 3-1-06 (0.43 mg , 1.68mmol) was dissolved in tetrahydrofuran (1mL), and slowly added dropwise to the reaction solution. After 10 minutes, add isoamyl nitrite (315.24mg, 2.69mmol). After the addition was completed, the reaction was maintained at 5°C for 1 hour. Use high-efficiency liquid The reaction was detected by phase mass spectrometry coupled with chromatography. After the reaction solution was quenched with saturated aqueous ammonium chloride solution, extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain the crude product of the title compound. 455.00mg.

The structural characterization data are as follows:

ESI-MS(m/z):285.0[M+H] ⁺ .

Step 7: Synthesis of N-(7-amino-4-chloro-3-fluoro-8-oxo-5,6,7,8-tetralin-1-yl)acetamide

Dissolve the crude product of compound 3-1-07 (0.40g, 1.41mmol) in methanol (10mL), then add 3mol/L hydrochloric acid aqueous solution (1mL) and 10% palladium on carbon (40.00mg). After the addition is completed, use hydrogen The reaction system was ball-displaced three times, and the reaction was carried out at room temperature for 1 hour under a hydrogen atmosphere, and the reaction was detected by high-performance liquid chromatography-mass spectrometry. The reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain 0.43 g of crude hydrochloride of the title compound.

The structural characterization data are as follows:

ESI-MS(m/z):271.0[M+H] ⁺ .

Step 8: (9H-fluoren-9-yl)methyl (8-acetamide-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetralin-2-yl)amino Synthesis of formate

The crude hydrochloride salt of compound 3-1-08 (0.43g, 1.19mmol) was dissolved in 1,4-dioxane (15mL), and then sodium bicarbonate (400.35mg, 4.77mmol) and water (5mL) were added ) and 9-fluorenylmethyl-N-succinimidyl carbonate (481.81 mg, 1.43 mmol). After completion, stir and react at room temperature for 2 hours. Use high-performance liquid chromatography-mass spectrometry to detect the reaction. The reaction solution was poured into water, and then extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by C18 reverse-phase column (acetonitrile: 0.05% formic acid in water = 20%-100%) to obtain 301.00 mg of the title compound.

The structural characterization data are as follows:

ESI-MS(m/z):493.2[M+H] ⁺ .

Step 9: (9H-fluoren-9-yl)methyl (8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetralin-2-yl)aminomethyl Synthesis of acid esters

Dissolve compound 3-1-09 (300.00mg, 608.61μmol) in dioxane (5mL), add 12mol/L concentrated hydrochloric acid (1mL), complete the addition, raise the temperature to 60°C and react for 2 hours, use high-efficiency liquid The reaction was detected by phase mass spectrometry coupled with chromatography. The reaction solution was poured into water, and then extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by flash silica gel column (ethyl acetate: petroleum ether = 0-50%) to obtain 198.00 mg of the title compound.

The structural characterization data are as follows:

ESI-MS(m/z):451.1[M+H] ⁺ .

Step 10: (9H-fluoren-9-yl)methyl ((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxy-2,3,9, 10,13,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolizino[1,2-b]quinolin-1-yl)amino Synthesis of formate

(S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolazine-3,6,10(4H)-trione (138.72 mg, 526.96 μmol) and compound 3-1-10 (198.00 mg, 439.13 μmol) were added to toluene (10 mL), and then p-toluenesulfonic acid (75.53 mg, 439.13 μmol) was added. After the addition was completed, the temperature was raised to 140°C for reaction 4 hours, the reaction solution was directly evaporated to dryness under reduced pressure at 140°C to obtain a crude product, which was purified by a flash silica gel column (methanol: dichloromethane = 0-5%) to obtain 256.00 mg of the title compound.

The structural characterization data are as follows:

ESI-MS(m/z):678.1[M+H] ⁺ .

Step 11: (1S,9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H- Benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione and (1R,9S)-1-amino- 4-Chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4' :Synthesis of 6,7]indolizino[1,2-b]quinoline-10,13-dione

Dissolve compound 3-1-11 (201.18 mg, 296.67 μmol) in N,N-dimethylformamide (4 mL), then add diethylamine (108.49 mg, 1.48 mmol), complete the addition, and react at room temperature for 0.5 hours. , using high-performance liquid chromatography-mass spectrometry to detect the reaction. After the ethylenediamine was evaporated under reduced pressure from the reaction solution, the pH was adjusted to 2-3 with 1 mol/L hydrochloric acid aqueous solution. The reaction solution was directly purified by preparative high performance liquid chromatography to obtain the title compound 3-1-A (44.00 mg). 3-1-B(43.00mg).

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

time[min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL/min] 0 10 90 28 3 10 90 28 18 70 30 28

3-1-A (6min LCMS peak is near the front, retention time: 1.276min)

The structural characterization data are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.00 (d, J = 10.3 Hz, 1H), 7.33 (s, 1H), 6.54 (s, 1H), 5.62 (d, J = 19.3 Hz, 1H) ,5.44(s,2H),5.38(d,J＝19.3Hz,1H),4.43-4.38(m,1H),3.28-3.10(m,2H),2.22-2.12(m,1H),2.12-2.02 (m,1H),1.93-1.80(m,2H),0.87(t,J=7.3Hz,3H).

ESI-MS(m/z):456.1[M+H] ⁺ .

3-1-B (6min LCMS peak appears later, retention time: 1.300min) structural characterization data are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ7.98 (d, J = 10.3 Hz, 1H), 7.32 (s, 1H), 5.61 (d, J = 19.4 Hz, 1H), 5.44 (s, 2H) ,5.32(d,J＝19.4Hz,1H),4.44-4.36(m,1H),3.33-3.25(m,1H),3.22-3.11(m,1H),2.23-2.13(m,1H),2.11 -2.03(m,1H),1.96-1.82(m,2H),0.89(t,J=7.3Hz,3H).

ESI-MS(m/z):456.1[M+H] ⁺ .

6min LCMS conditions:

Column: Waters SunFire C18 OBD 4.6mm×50mm×5.0μm

Mobile phase A: 0.05% acetonitrile; mobile phase B: water (0.05% formic acid)

time[min] Mobile phase A[%] Mobile phase B [%] Flow rate [mL/min] 0 90 10 2 4.2 10 90 2 5.7 10 90 2 5.71 90 10 2 6.70 90 10 2

Example 9 N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-six Hydrogen-1H,12H-benzo[de]pyrano[3',4:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide and N -((1R,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-Benzo[de]pyrano[3',4:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide

Step 1: (S)-10-benzyl-23-(2-(methanesulfonyl)pyrimidin-5-yl)-6,9,12,15,18-pentaoxo-3-oxo-5 ,Synthesis of 8,11,14,17-pentaazaoctadecane-22-ynecarboxylic acid

Compound 3-4-01 (30.00 mg, 70.00 μmol) was dissolved in N,N-dimethylformamide (1 mL), and 2,5-dioxopyrrolidin-1-yl 6-(2-(methyl Sulfonic acid) pyrimidin-5-yl) hexyl-5-ynamide (28.00 mg, 77.00 μmol), react at room temperature for 1 hour, and monitor the reaction with high-performance liquid chromatography-mass spectrometry. The reaction solution was directly purified by preparative high performance liquid chromatography, and the prepared solution was freeze-dried to obtain the title compound 3-4-03 (20.00 mg).

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

time[min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL/min] 0.00 10 90 28 2.00 10 90 28 18.00 90 10 28

The structural characterization data are as follows:

ESI-MS(m/z):691.0[M+18] ⁺ .

Step 2: N-((S)-10-benzyl-1-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2 ,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline -1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecane-16-yl)-6-( 2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-amide and N-((S)-10-benzyl-1-((1R,9S)-4-chloro-9-ethyl- 5-Fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4': 6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11, Synthesis of 14-tetraazahexadecane-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-amide

Single configuration compound 3-1-A (36.00 mg, 79.70 μmol) and compound 3-4-03 (64.43 mg, 95.64 μmol) were dissolved in N,N-dimethylformamide (2 mL), and then added 4-(4,6-Dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (46.98 mg, 159.40 μmol) and triethylamine (24.19 mg, 239.10 μmol), after addition, The reaction was carried out at room temperature for 1 hour, and the reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was directly purified by high performance liquid chromatography to obtain the title compound 3-4-04-A (51.00 mg) in a single configuration.

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

time[min] Mobile phase A[%] Mobile phase B [%] Flow rate [mL/min] 0 30 70 28 3 30 70 28 18 90 10 28

The structural characterization data are as follows:

ESI-MS(m/z):1111.0[M+H] ⁺ .

Single configuration compound 3-1-B (36.00 mg, 79.70 μmol) and compound 3-4-03 (64.43 mg, 95.64 μmol) were dissolved in N,N-dimethylformamide (2 mL), and then added 4-(4,6-Dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (46.98 mg, 159.40 μmol) and triethylamine (24.19 mg, 239.10 μmol), after addition, The reaction was carried out at room temperature for 1 hour, and the reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was directly purified by high performance liquid chromatography to obtain the title compound 3-4-04-B (52.00 mg) in a single configuration.

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

time[min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL/min] 0 30 70 28 3 30 70 28 18 90 10 28

The structural characterization data are as follows:

ESI-MS(m/z):1111.0[M+H] ⁺ .

Step 3: N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-six Hydrogen-1H,12H-benzo[de]pyrano[3',4:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide and N -((1R,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H, Synthesis of 12H-benzo[de]pyrano[3',4:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide

Weigh compound 3-4-04-A (40.00 mg, 35.99 μmol) and dissolve it in a mixed solvent of dichloromethane (2 mL) and methanol (1 mL). Then add 4 mol/L hydrochloric acid ethyl acetate (1 mL) and complete the addition. , react at room temperature for 0.5 hours, and detect the reaction with high-performance liquid chromatography-mass spectrometry. The reaction solution was directly concentrated to dryness under reduced pressure to obtain a crude product, which was purified by high performance liquid chromatography to obtain the title compound 3-4-A (4.75 mg) in a single configuration.

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

time[min] Mobile phase A [%] Mobile phase B[%] Flow rate [mL/min] 0 15 85 28 3 15 85 28 18 90 10 28

The structural characterization data are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.50 (d, J = 8.9 Hz, 1H), 8.05 (d, J = 10.3 Hz, 1H), 7.33 (s, 1H), 6.55 (s, 1H) ,5.67-5.60(m,1H),5.49(t,J＝5.8Hz,1H),5.43(s,2H),5.21(s,2H),3.96(d,J＝5.8Hz,2H),3.32- 3.22(m,2H),2.28-2.15(m,2H),1.93-1.80(m,2H),0.87(t,J=7.3Hz,3H).

ESI-MS(m/z):514.0[M+H] ⁺ .

Weigh compound 3-4-04-B (40.00 mg, 35.99 μmol) and dissolve it in a mixed solvent of dichloromethane (2 mL) and methanol (1 mL). Then add 4 mol/L hydrochloric acid ethyl acetate (1 mL) and complete the addition. , react at room temperature for 0.5 hours, and detect the reaction with high-performance liquid chromatography-mass spectrometry. The reaction solution was directly concentrated to dryness under reduced pressure to obtain a crude product, which was purified by high performance liquid chromatography to obtain the title compound 3-4-B (8.24 mg) in a single configuration.

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

time[min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL/min] 0 15 85 28 3 15 85 28 18 90 10 28

The structural characterization data are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.52 (d, J = 9.0 Hz, 1H), 8.05 (d, J = 10.3 Hz, 1H), 7.34 (s, 1H), 6.55 (s, 1H) ,5.68-5.58(m,1H),5.53(t,J＝5.8Hz,1H),5.43(d,J＝2.9Hz,2H),5.20(d,J＝7.3Hz,2H),3.97(d, J＝5.7Hz,2H),3.31-3.21(m,2H),2.26-2.15(m,2H),1.92-1.82(m,2H),0.87(t,J＝7.3Hz,3H).

ESI-MS(m/z):514.0[M+H] ⁺ .

Example 10 (S)-N-(2-(4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro- 1H-pyran[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)-2-hydroxy-N-isopropylacetamide

Step 1: Synthesis of 1-(4-fluoro-3-methylphenyl)-3-(isopropylamine)propan-1-one

Dissolve compound 4-12-01 (500.00 mg, 3.29 mmol), formaldehyde aqueous solution (2.5 mL, 37%) and isopropylamine (388.46 mg, 6.57 mmol) in isopropanol (5 mL) at 20° C. Concentrated hydrochloric acid (2.5 mL) was added dropwise, the reaction solution was stirred at 100°C for 16 hours, and the reaction was detected by high-performance liquid chromatography-mass spectrometry. The reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was purified by preparative high performance liquid chromatography. The preparation solution was freeze-dried to obtain 200.00 mg of the title compound.

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

time[min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL/min] 0.00 10 90 28 2.00 10 90 28 18.00 90 10 28

The structural characterization data are as follows:

ESI-MS(m/z):224.1[M+H] ⁺ .

Step 2: Synthesis of 1-(4-fluoro-5-methyl-2-nitrophenyl)-3-(isopropylamine)propan-1-one

At 0°C, compound 4-12-02 (100.00mg, 0.49mmol) was added to concentrated sulfuric acid (0.5mL), and potassium nitrate (54.34mg, 0.54mmol) was added. The reaction solution was kept at 0°C for 1 hour. Use high-efficiency liquid The reaction was detected by phase mass spectrometry coupled with chromatography. The reaction solution was poured into ice water, and purified by reverse phase column (acetonitrile: 0.05% formic acid in water = 0-30%) to obtain 90.00 mg of the title compound.

The structural characterization data are as follows:

ESI-MS(m/z):269.0[M+H] ⁺ .

Step 3: Synthesis of 1-(2-amino-4-fluoro-5-methylphenyl)-3-(isopropylamine)propan-1-one

At 25°C, compound 4-12-03 (200.00mg, 0.75mmol) was added to methanol (20.0mL), 10% palladium on carbon (10.00mg) was added, the reaction solution was replaced with hydrogen, and the reaction was maintained at 20°C in a hydrogen environment. After 16 hours, the reaction was detected by high-performance liquid chromatography-mass spectrometry. The reaction solution was filtered and concentrated under reduced pressure to obtain 183.00 mg of the title compound.

The structural characterization data are as follows:

ESI-MS(m/z):239.1[M+H] ⁺ .

Step 4: (S)-4-ethyl-8-fluoro-4-hydroxy-11-(2-(isopropylamino)ethyl)-9-methyl-1,12-dihydro-14H-pyran Synthesis of [3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione

Compound 4-12-04 (50.00 mg, 0.21 mmol) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f ] Indolazine-3,6,10(4H)-trione (55.23mg, 0.21mmol) was added to toluene (3mL), p-toluenesulfonic acid (3.61mg, 0.02mmol) was added, and the reaction solution was reacted at 130°C for 4 hours , using high-performance liquid chromatography-mass spectrometry to detect the reaction. The reaction solution was concentrated under reduced pressure, and the crude product was purified by preparative high-performance liquid chromatography. The prepared solution was freeze-dried to obtain 2.00 mg of the trifluoroacetate salt of the title compound.

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% trifluoroacetic acid)

time[min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL/min] 0.00 8 92 28 2.00 8 92 28 18.00 60 40 28

The structural characterization data are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.58 (s, 2H), 8.22 (d, J = 8.1Hz, 1H), 7.97 (d, J = 10.7Hz, 1H), 7.35 (s, 1H) ,6.59(s,1H),5.47(s,2H),5.41(s,2H),3.58-3.45(m,3H),3.31-3.23(m,2H),2.56(s,3H),1.98-1.80 (m,2H),1.26(d,J＝6.3Hz,6H),0.89(t,J＝7.3Hz,3H).

ESI-MS(m/z):466.2[M+H] ⁺

Step 5: (S)-2-((tert-butyldiphenylsilyl)oxy)-N-(2-(4-ethyl-8-fluoro-4-hydroxy-9-methyl-3 ,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-11- Synthesis of ethyl)-N-isopropylacetamide

Compound 4-12-05 (22.00 mg, 47.26 μmol) and 2-((tert-butyldiphenylsilyl)oxy)acetic acid (16.35 mg, 51.99 μmol) were dissolved in N,N-dimethylform amide (1mL), then add HATU (21.55mg, 56.71μmol) and N,N-diisopropylethylamine (18.32mg, 141.78μmol), complete the addition, react at room temperature for 0.5 hours, and use high performance liquid chromatography mass spectrometry Chromatographic detection reaction. The reaction solution was directly purified through a C18 reverse-phase column (acetonitrile: 0.05% formic acid aqueous solution = 30%-100%) to obtain 18.00 mg of the title compound.

The structural characterization data are as follows:

ESI-MS(m/z):762.3[M+H] ⁺ .

Step six: (S)-N-(2-(4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro- Synthesis of 1H-pyran[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)-2-hydroxy-N-isopropylacetamide

Compound 4-12-06 (18.00 mg, 23.62 μmol) was dissolved in N,N-dimethylformamide (1 mL), then potassium fluoride (6.86 mg, 118.12 μmol) was added, and the temperature was raised to 50°C for reaction. 1 hour, and the reaction was detected using high-performance liquid chromatography-mass spectrometry. The reaction solution was directly purified by high performance liquid chromatography to obtain 1.53 mg of the title compound.

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

time[min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL/min] 0.00 15 85 28 18.00 90 10 28

The structural characterization data are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.53(d,J＝8.2Hz,1H),7.91(d,J＝10.8Hz,1H),7.32(s,1H),6.55(s,1H) ,5.44(d,J＝13.8Hz,4H),4.72(t,J＝5.5Hz,1H),4.21(d,J＝5.5Hz,2H),3.99-3.90(m,1H),3.54-3.38( m,4H),2.54(s,3H),1.92-1.83(m,2H),1.17(dd,J＝6.6,3.1Hz,6H),0.87(t,J＝7.3Hz,3H).

ESI-MS(m/z):524.2[M+H] ⁺ .

Example 11 (S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxy-3,4,12,14-tetrahydro-1H -Pyran[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-1-hydroxycyclopropanecarboxamide

The raw material (S)-11-(aminomethyl)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-1H-pyran[3',4':6,7]indolazine Para[1,2-b]quinoline-3,14(4H,12H)-dione (4-10-01, prepared according to the synthesis method of patent WO2020219287, 30.00 mg, 67.00 μmol), 1-hydroxycyclopropanecarboxylic acid (7.56 mg, 0.074 mmol) was dissolved in DMF (1 mL), HBTU (34.30 mg, 0.14 mmol) and diisopropylethylamine (26.09 mg, 0.20 mmol) were added with stirring, and the reaction was carried out at room temperature for 4 hours. Add water and ethyl acetate, stir, and let stand for liquid separation. The organic phase is washed with saturated brine and concentrated under reduced pressure. The concentrate was purified by preparative thin layer chromatography (dichloromethane:methanol=20:1), and then purified by preparative high performance liquid chromatography to obtain 1.20 mg of solid.

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

time[min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL/min]

0.00 20 80 28 2.00 20 80 28 18.00 80 20 28

The structural characterization data are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.96 (t, J = 6.0 Hz, 1H), 8.51 (d, J = 8.0 Hz, 1H), 7.90 (d, J = 10.8 Hz, 1H), 7.31 (s,1H),6.53(s,1H),6.30(s,2H),6.30(s,1H),5.52(s,2H),5.44(s,2H),4.84(d,J＝6.0Hz, 2H),2.51(s,3H),1.91-1.81(m,2H),1.01(dd,J＝7.2,4.1Hz,2H),0.87(t,J＝7.3Hz,3H),0.83(t,J =3.6Hz,2H).

ESI-MS(m/z):494.1[M+1] ⁺ .

Example 12 (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-1,2,3,9,12,15-hexahydro- 10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione and (1R,9S)- 1-amino-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyridine Prando[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione

Step 1: Synthesis of (E)-4-(5-acetylamino-3-fluoro-2-methylphenyl)-2-methyl-3-butenoic acid

Weigh N-(3-bromo-5-fluoro-4-methylphenyl)acetamide (2.00g, 8.13mmol) and 2-methyl-3-butenoic acid (976.44mg, 9.75mmol) and dissolve them in 1 , into a mixed solvent of 4-dioxane (15mL) and water (5mL), then add tris(o-tolyl)phosphine (247.37mg, 812.76μmol), palladium acetate (91.24mg, 406.38μmol) and N,N -Diisopropylethylamine (2.31g, 17.88mmol) was added, and the reaction system was replaced with nitrogen three times, and the temperature was raised to 80°C for 3 hours under a nitrogen atmosphere. The reaction was detected by high-performance liquid chromatography-mass spectrometry. After the reaction solution was cooled to room temperature, 1 mol/L sodium hydroxide aqueous solution (60 mL) and ethyl acetate (50 mL) were added and the mixture was separated by shaking. After separating the lower aqueous phase, adjust the pH to about 3 with 4 mol/L hydrochloric acid aqueous solution, and then extract with ethyl acetate. The combined organic phases are washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated under reduced pressure to obtain the title Compound 1.90g.

The structural characterization data are as follows:

ESI-MS(m/z):266.1[M+H] ⁺ .

Step 2: Synthesis of 4-(5-acetylamino-3-fluoro-2-methylphenyl)-2-methylbutyric acid

Dissolve (E)-4-(5-acetamido-3-fluoro-2-methylphenyl)-2-methyl-3-butenoic acid (1.90g, 7.16mmol) in methanol (40mL), 10% palladium on carbon (0.15g) was added under nitrogen protection, and then the reaction system was replaced three times with a hydrogen balloon, and the reaction was carried out under a hydrogen atmosphere for 2 hours, and the reaction was detected by high-performance liquid chromatography-mass spectrometry. The reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain 1.51 g of the title compound.

The structural characterization data are as follows:

ESI-MS(m/z):268.1[M+H] ⁺ .

Step 3: Synthesis of N-(3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetralin-1-yl)acetamide

Weigh 4-(5-acetamido-3-fluoro-2-methylphenyl)-2-methylbutyric acid (1.50g, 5.61mmol) and dissolve it in trifluoroacetic acid (20mL), then cool to 5°C. Trifluoroacetic anhydride (2.36g, 11.22mmol) was added dropwise. After the addition was completed, the reaction was maintained at 5°C for 2 hours, and the reaction was detected by high-performance liquid chromatography-mass spectrometry. Slowly pour the reaction solution into a saturated sodium bicarbonate aqueous solution, then extract with ethyl acetate, combine the organic phases, wash with saturated brine, dry the organic phase with anhydrous sodium sulfate, filter, and evaporate the filtrate to dryness under reduced pressure to obtain a crude product. The crude product was purified by flash silica gel column (ethyl acetate: petroleum ether = 0-30%) to obtain 1.05 g of the title compound.

The structural characterization data are as follows:

ESI-MS(m/z):250.1[M+H] ⁺ .

Step 4: Synthesis of N-(7-bromo-3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetralin-1-yl)acetamide

Weigh N-(3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetralin-1-yl)acetamide (0.55g, 2.21mmol) and dissolve it in acetic acid (8mL), then add bromine (387.85mg, 2.43mmol), complete the addition, raise the temperature to 50°C and react for 15 hours, and detect the reaction with high performance liquid chromatography mass spectrometry. The reaction solution was directly evaporated to dryness under reduced pressure to obtain a crude product, which was purified by a flash silica gel column (ethyl acetate: petroleum ether = 0-30%) to obtain 461.00 mg of the title compound.

The structural characterization data are as follows:

ESI-MS(m/z):328.0[M+H] ⁺ .

Step 5: Synthesis of N-(7-azido-3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetralin-1-yl)acetamide

Weigh N-(7-bromo-3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetralin-1-yl)acetamide (460.00mg, 1.40mmol) Dissolve in N,N-dimethylformamide (10 mL), then add sodium azide (273.37 mg, 4.21 mmol), complete the addition, and react at room temperature for 1 hour. Use high-performance liquid chromatography-mass spectrometry to detect the reaction. Slowly pour the reaction solution into water, then extract with ethyl acetate, combine the organic phases, wash with saturated brine, dry the organic phase with anhydrous sodium sulfate, filter, and evaporate the filtrate to dryness under reduced pressure to obtain a crude product, which is purified by a fast silica gel column (Ethyl acetate:petroleum ether=0-50%), 347.00 mg of the title compound was obtained.

The structural characterization data are as follows:

ESI-MS(m/z):291.1[M+H] ⁺ .

Step 6: Synthesis of N-(7-amino-3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetralin-1-yl)acetamide

Weigh N-(7-azido-3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetralin-1-yl)acetamide (347.00 mg, 1.20 mmol) in tetrahydrofuran (10 mL), add 10% palladium on carbon (30.00 mg) under nitrogen protection, then replace the reaction system with a hydrogen balloon three times, and react under a hydrogen atmosphere for 2 hours, and detect with high-performance liquid chromatography-mass spectrometry. reaction. The reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain a crude product, which was purified by a C18 reverse-phase column (acetonitrile: 0.05% formic acid aqueous solution = 0%-30%) to obtain 205.00 mg of the title compound.

The structural characterization data are as follows:

ESI-MS(m/z):265.1[M+H] ⁺ .

Step 7: (9H-fluoren-9-yl)methyl(8-acetylamino-6-fluoro-2,5-dimethyl-1-oxo-1,2,3,4-tetralin-2 -Synthesis of carbamate

Weigh N-(7-amino-3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetralin-1-yl)acetamide (200.00mg, 756.73μmol) Dissolve in a mixed solvent of 1,4-dioxane (6mL) and water (3mL), then add sodium bicarbonate (254.28mg, 3.03mmol) and 9-fluorenylmethyl-N-succinimidyl carbonate After adding the ester (650.55 mg, 1.14 mmol), stir and react at room temperature for 2 hours, and detect the reaction with high-performance liquid chromatography-mass spectrometry. Slowly pour the reaction solution into water, and then extract with ethyl acetate. Combine the organic phases, wash with saturated brine, dry the organic phase over anhydrous sodium sulfate, filter, and evaporate the filtrate to dryness under reduced pressure to obtain a crude product. The crude product is passed through C18 reverse phase. Column purification (acetonitrile: 0.05% formic acid aqueous solution = 20%-80%) gave 301.00 mg of the title compound.

The structural characterization data are as follows:

ESI-MS(m/z):487.0[M+H] ⁺ .

Step 8: (9H-fluoren-9-yl)methyl(8-amino-6-fluoro-2,5-dimethyl-1-oxo1,2,3,4-tetralin-2-yl )Synthesis of urethane

(9H-Fluoren-9-yl)methyl(8-acetamido-6-fluoro-2,5-dimethyl-1-oxo-1,2,3,4-tetralin-2-yl ) Carbamate (101.00 mg, 207.59 μmol) was dissolved in 1,4-dioxane (5 mL), and then 3 mol/L hydrochloric acid aqueous solution (5 mL) was added. After the addition was completed, the temperature was raised to 50°C and reacted for 15 hours. High-performance liquid chromatography-mass spectrometry coupled to chromatography detects the reaction. Slowly pour the reaction solution into a saturated sodium bicarbonate aqueous solution, and then extract with ethyl acetate. The combined organic phases are washed with saturated brine, the organic phase is dried over anhydrous sodium sulfate, filtered, and the filtrate is evaporated to dryness under reduced pressure to obtain a crude product. Fast silica gel column purification (methanol: dichloromethane = 0%-5%) gave 71.00 mg of the title compound.

The structural characterization data are as follows:

ESI-MS(m/z):445.2[M+H] ⁺ .

Step 9: (9H-fluoren-9-yl)methyl ((9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-2, 3,9,10,13,15–Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline- Synthesis of 1-yl)carbamate

(9H-Fluoren-9-yl)methyl(8-amino-6-fluoro-2,5-dimethyl-1-oxo1,2,3,4-tetralin-2-yl)amino Formate (35.00 mg, 132.96 μmol) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolazine-3,6 , 10(4H)-triketone (49.25mg, 110.80μmol) was added to toluene (3mL), and then p-toluenesulfonic acid (19.08mg, 110.80μmol) was added. After the addition was completed, the temperature was raised to 140°C for 4 hours. The reaction solution was directly The crude product was obtained by evaporating to dryness under reduced pressure at 140°C. The crude product was purified by C18 reverse-phase column (acetonitrile: 0.05% formic acid aqueous solution = 20%-80%) to obtain 21.00 mg of the title compound.

The structural characterization data are as follows:

ESI-MS(m/z):672.2[M+H] ⁺ .

Step 10: (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-1,2,3,9,12,15-hexahydro-10H ,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione and (1R,9S)-1 -Amino-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyran Synthesis of [3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione

(9H-Fluoren-9-yl)methyl((9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-2,3, 9,10,13,15–Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-1- base) carbamate (21.00 mg, 31.26 μmol) was dissolved in N,N-dimethylformamide (1 mL), then diethylamine (0.2 mL) was added, the addition was completed, and the reaction was carried out at room temperature for 0.5 hours. Use high-efficiency liquid The reaction was detected by phase mass spectrometry coupled with chromatography. After the ethylenediamine was evaporated under reduced pressure from the reaction solution, the pH was adjusted to 2-3 with 1 mol/L hydrochloric acid aqueous solution. The reaction solution was directly purified by preparative high performance liquid chromatography to obtain two isomers 5-13-A (1.30 mg) and 5-13-B (1.68 mg).

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

time[min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL/min] 0.00 10 90 28 3.00 10 90 28 18.00 90 10 28

The structural characterization data of 5-13-A (6min LCMS peak is near the front, retention time: 1.373min) are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ7.88 (d, J = 10.6 Hz, 1H), 7.36 (s, 1H), 6.58 (s, 1H), 5.60 (d, J = 3.5 Hz, 2H) ,5.46(d,J＝2.5Hz,2H),3.25-3.17(m,2H),2.41(s,3H),2.38-2.28(m,2H),1.91-1.84(m,2H),1.79(s ,3H),0.88(t,J=7.3Hz,3H).

ESI-MS(m/z):450.2[M+H] ⁺ .

The structural characterization data of 5-13-B (6min LCMS peak is later, retention time: 1.523min) are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ7.76 (d, J = 10.8 Hz, 1H), 7.30 (s, 1H), 6.52 (s, 1H), 5.73 (d, J = 19.8 Hz, 1H) ,5.50-5.40(m,3H),3.26-3.17(m,1H),3.08-2.96(m,1H),2.38(s,3H),2.19-2.11(m,1H),2.04(td,J＝ 13.0,5.1Hz,1H),1.91-1.79(m,2H),1.34(s,3H),0.87(t,J＝7.3Hz,3H).

ESI-MS(m/z):450.2[M+H] ⁺ .

6min LCMS conditions:

Column: Waters SunFire C18 OBD 4.6mm×50mm×5.0μm

Mobile phase A: 0.05% acetonitrile; mobile phase B: water (0.05% formic acid)

time[min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL/min] 0 90 10 2 4.2 10 90 2 5.7 10 90 2 5.71 90 10 2 6.70 90 10 2

Example 13 (1S,9S)-1-(aminomethyl)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydrogen -10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione and (1R,9S) -1-(Aminomethyl)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de ]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione

Step 1: N-(7-((dimethylamino)methylene)-3-fluoro-4-methyl-8-oxy-5,6,7,8-tetralin-1-yl)ethyl Synthesis of amides

Compound 5-13-04 (1.00g, 4.25mmol) was dissolved in N,N-dimethylformamide dimethyl acetal (10mL), then the temperature was raised to 120°C and reacted for 3 hours. High performance liquid chromatography mass spectrometry was used. Detect the reaction using chromatography. After the reaction solution was cooled to room temperature, it was directly evaporated to dryness under reduced pressure to obtain a crude product. The crude product was purified by a flash silica gel column (ethyl acetate: petroleum ether = 20%-100%) to obtain 891.00 mg of the title compound.

The structural characterization data are as follows:

ESI-MS(m/z):291.1[M+H] ⁺ .

Step 2: Synthesis of N-(7-(aminomethylene)-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetralin-1-yl)acetamide

Dissolve compound 5-7-01 (0.89g, 3.07mmol) in ethanol (25mL), then add ammonium acetate (2.36g, 30.65mmol), complete the addition, react at room temperature for 16 hours, and use HPLC-MS chromatography Detect the reaction. The reaction solution solvent was evaporated to dryness under reduced pressure, then dichloromethane (30 mL) and water (20 mL) were added, stirred and allowed to stand to separate the organic phase, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain the title compound. 785.00mg.

The structural characterization data are as follows:

ESI-MS(m/z):263.1[M+H] ⁺ .

Step 3: Synthesis of N-(7-(aminomethyl)-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetralin-1-yl)acetamide

Dissolve compound 5-7-02 (0.80g, 3.05mmol) in ethanol (200mL), then add 10% palladium on carbon (0.40mg) and concentrated hydrochloric acid (0.2mL). After the addition is completed, replace the reaction system with a hydrogen balloon three times. , then react at room temperature for 3 hours under a hydrogen atmosphere, and detect the reaction with high-performance liquid chromatography-mass spectrometry. The reaction solution was directly filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain 905.00 mg of the hydrochloride salt of the title compound.

The structural characterization data are as follows:

ESI-MS(m/z):265.1[M+H] ⁺ .

Step 4: (9H-fluoren-9-yl)methyl((8-acetamide-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetralin-2-yl) )Synthesis of methyl)carbamate

Dissolve the hydrochloride of compound 5-7-03 (0.90g, 2.99mmol) in 1,4-dioxane (20mL), then add sodium bicarbonate (1.01g, 11.97mmol) and water (10mL) and 9-fluorenylmethyl-N-succinimidyl carbonate (1.21g, 3.59mmol). After completion, stir and react at room temperature for 1 hour. Use high-performance liquid chromatography-mass spectrometry to detect the reaction. The reaction solution was poured into water, and then extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was dried under reduced pressure to obtain a crude product. The crude product was purified by flash silica gel column (ethyl acetate: petroleum ether = 0-50%) to obtain 1.30 g of the title compound.

The structural characterization data are as follows:

ESI-MS(m/z):487.1[M+H] ⁺ .

Step 5: (9H-fluoren-9-yl)methyl ((8-amino-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetralin-2-yl) Synthesis of methyl)carbamate

Dissolve compound 5-7-04 (0.80g, 1.64mmol) in 1,4-dioxane (20mL), add 3mol/L hydrochloric acid aqueous solution (20mL) under nitrogen protection, complete the addition, and raise the temperature to 60°C for reaction. After 15 hours, the reaction was detected by high-performance liquid chromatography-mass spectrometry. The reaction solution was slowly poured into water, and then extracted with ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was evaporated to dryness under reduced pressure to obtain crude product. The crude product was purified by flash silica gel column (ethyl acetate: petroleum ether = 0-40%) to obtain 561.00 mg of the title compound.

The structural characterization data are as follows:

ESI-MS(m/z):445.1[M+H] ⁺ .

Step six: (9H-fluoren-9-yl)methyl (((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3, 9,10,13,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-1- Synthesis of methyl)carbamate

(S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolazine-3,6,10(4H)-trione (597.00 mg, 2.27mmol) and compound 5-7-05 (840.00mg, 1.89mmol) were added to toluene (60mL), and then p-toluenesulfonic acid (325.00mg, 1.89mmol) was added. After the addition was completed, the temperature was raised to 140°C for reaction 4 hours, and then the reaction solution was directly evaporated to dryness under reduced pressure at 140°C to obtain the crude product. The crude product was purified by flash silica gel column (methanol: dichloromethane = 0-5%) to obtain 563.00 mg of the title compound.

The structural characterization data are as follows:

ESI-MS(m/z):672.2[M+H] ⁺ .

Step 7: (1S,9S)-1-(aminomethyl)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro- 10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione and (1R,9S)- 1-(aminomethyl)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de] Synthesis of pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione

Compound 5-7-06 (454.00 mg, 675.89 μmol) was dissolved in N,N-dimethylformamide (5 mL), then diethylamine (1 mL) was added, and the reaction was completed at room temperature for 0.5 hours. Use high-performance liquid phase Mass spectrometry coupled to chromatography detects the reaction. After the ethylenediamine was evaporated under reduced pressure from the reaction solution, the pH was adjusted to 2-3 with formic acid, and the reaction solution was directly purified by preparative high-performance liquid chromatography. The preparation solution was freeze-dried to obtain the title compound 5-7-A (32.00 mg). and 5-7-B (56.00mg).

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

time[min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL/min] 0 10 90 28 3 10 90 28 18 90 10 28

The structural characterization data of 5-7-A (6min LCMS peak is near the front, retention time: 1.488min) are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ7.88 (d, J = 10.6 Hz, 1H), 7.36 (s, 1H), 6.58 (s, 1H), 5.60 (d, J = 3.5 Hz, 2H) ,5.46(d,J＝2.5Hz,2H),3.25-3.17(m,2H),2.41(s,3H),2.38-2.28(m,2H),1.91-1.84(m,2H),1.79(s ,3H),0.88(t,J=7.3Hz,3H).

ESI-MS(m/z):450.2[M+H] ⁺ .

The structural characterization data of 5-7-B (6min LCMS peak is later, retention time: 1.596min) are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ7.76 (d, J = 10.8 Hz, 1H), 7.30 (s, 1H), 6.52 (s, 1H), 5.73 (d, J = 19.8 Hz, 1H) ,5.50-5.40(m,3H),3.26-3.17(m,1H),3.08-2.96(m,1H),2.38(s,3H),2.19-2.11(m,1H),2.04(td,J＝ 13.0,5.1Hz,1H),1.91-1.79(m,2H),1.34(s,3H),0.87(t,J＝7.3Hz,3H).

ESI-MS(m/z):450.2[M+H] ⁺ .

6min LCMS conditions:

Column: Waters SunFire C18 OBD 4.6mm×50mm×5.0μm

Mobile phase A: 0.05% acetonitrile; mobile phase B: water (0.05% formic acid)

time[min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL/min] 0 90 10 2 4.2 10 90 2 5.7 10 90 2 5.71 90 10 2 6.70 90 10 2

Example 14 N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-2,3,9,10, 13,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1 -Hydroxycyclopropane-1-carboxamide or N-((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-2, 3,9,10,13,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline- 1-yl)-1-hydroxycyclopropane-1-carboxamide

Step 1: 1-((tert-butyldiphenylsilyl)oxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl -10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazine And[1,2-b]quinolin-1-yl)cyclopropane-1-carboxamide or 1-((tert-butyldiphenylsilyl)oxy)-N-((1R,9S)- 9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo Synthesis of [de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)cyclopropane-1-carboxamide

A single configuration of compound 5-13-A (10.00 mg, 22.25 μmol) and 1-((tert-butyldiphenylsilyl)oxy)cyclopropane-1-carboxylic acid (11.36 mg, 33.37 μmol) Dissolve in N,N-dimethylformamide (1mL), then add HATU (12.68mg, 33.37μmol) and N,N-diisopropylethylamine (8.63mg, 66.74μmol), complete the addition, and react at room temperature 0.5 hours, and the reaction was detected by high-performance liquid chromatography-mass spectrometry. The reaction solution was directly purified through a C18 reverse-phase column (acetonitrile: 0.05% formic acid aqueous solution = 30%-100%) to obtain a single title compound 5-16-01-A (7 mg).

The structural characterization data are as follows:

ESI-MS(m/z):772.3[M+H] ⁺ .

Step 2: N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-2,3,9,10,13 ,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1- Hydroxycyclopropane-1-carboxamide or N-((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-2,3 ,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-1 Synthesis of -1-hydroxycyclopropane-1-carboxamide

Compound 5-16-01-A (7.00 mg, 9.07 μmol) was dissolved in N,N-dimethylformamide (1 mL), and then potassium fluoride (2.63 mg, 45.34 μmol) was added. After addition, the temperature was raised to 50 The reaction was carried out for 1 hour at ℃, and the reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was directly purified by high performance liquid chromatography to obtain the single title compound 5-16-A (1.73 mg).

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

time[min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL/min] 0.00 10 90 28 18.00 90 10 28

The structural characterization data are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.26 (s, 1H), 7.78 (d, J = 10.8Hz, 1H), 7.30 (s, 1H), 6.56 (s, 1H), 6.52 (s, 1H),5.52(d,J＝19.3Hz,1H),5.43(d,J＝4.4Hz,2H),4.94(d,J＝19.2Hz,1H),3.30-3.24(m,1H),3.11- 3.00(m,1H),2.95-2.84(m,1H),2.39(s,3H),1.98-1.80(m,3H),1.62(s,3H),0.87(t,J＝7.9Hz,3H) .

ESI-MS(m/z):534.2[M+H] ⁺ .

Example 15 N-((10S)-10-benzyl-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxy Generation-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4';6,7]indolizino[1,2-b ]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxo-5,8,11,14-tetraazahexadecane-16-yl)- 6-(2-(methanesulfonyl)pyrimidin-5-yl)hexadecylamide or N-((10S)-10-benzyl-1-(((1R,9S)-5-chloro-9-ethyl) Base-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3', 4';6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxo-5,8 ,11,14-tetraazahexadecane-16-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hexadecylamide

Step 1: (9S)-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo Isolation and purification of pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione

Compound 2-23 (16.00 mg) was purified by preparative high performance liquid chromatography, and two diastereomers were separated under the following purification conditions to obtain 5.10 mg of the trifluoroacetate salt of 2-23-A (retention time 9.85 min) and 7.12 mg of 2-23-B trifluoroacetate (retention time 10.62 min).

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% trifluoroacetic acid)

time[min] Mobile phase A [%] Mobile phase B[%] Flow rate [mL/min] 0 5 95 28

2 5 95 28 18 50 50 28

The structural characterization data are as follows:

2-23-A:

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.42(s,3H),8.27(s,1H),7.36(s,1H),6.59(s,1H),5.78-5.63(m,1H), 5.50-5.36(m,3H),5.10-5.06(m,1H),3.20-3.04(m,2H),2.56(s,3H),2.26-2.13(m,2H),1.93-1.79(m,2H ),0.88(t,J=7.2Hz,3H).

ESI-MS(m/z):452.1[M+H] ⁺ .

2-23-B：

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.42(s,3H),8.27(s,1H),7.36(s,1H),6.58(s,1H),5.78-5.63(m,1H), 5.50-5.36(m,3H),5.10-5.06(m,1H),3.20-3.04(m,2H),2.55(s,3H),2.26-2.13(m,2H),1.93-1.79(m,2H ),0.88(t,J=7.2Hz,3H).

ESI-MS(m/z):452.0[M+H] ⁺ .

Step 2: N-((10S)-10-benzyl-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo -2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4';6,7]indolizino[1,2-b] Quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxo-5,8,11,14-tetraazahexadecane-16-yl)-6 -(2-(methanesulfonyl)pyrimidin-5-yl)hexadecylamide or N-((10S)-10-benzyl-1-(((1R,9S)-5-chloro-9-ethyl) -9-Hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4 '6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxo-5,8, Synthesis of 11,14-tetraazahexadecane-16-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hexadecylamide

Dissolve the trifluoroacetate salt of 2-23-A (34.71 mg, 61.43 μmol) in N,N-dimethylformamide (1 mL) at 25°C, and add 3-4-03 (49.66 mg, 73.72μmol), HATU (35.01mg, 92.14μmol) and N,N-diisopropylethylamine (23.82mg, 184.29μmol). Keep the reaction at 25°C for 0.5 hours. Use high performance liquid chromatography-mass spectrometry to monitor the reaction. After the reaction solution was purified by preparative high performance liquid chromatography (conditions are as follows), the preparation solution was freeze-dried to obtain 11.04 mg of the title compound D-L-15, with a retention time of 7.5 min.

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

time[min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL/min] 0 30 70 28 3 30 70 28 18 90 10 28

The structural characterization data are as follows:

D-L-15：

ESI-MS(m/z):1107.3[M+H] ⁺ .

Example 16 N-((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxy-2,3,9,10,13,15-hexahydrogen -1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-cyclopropyl-2 -Hydroxyacetamide

Step 1: N-((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxy-2,3,9,10,13,15-hexahydro- 1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-cyclopropyl-2- Synthesis of hydroxyacetamide

The formate salt of compound 3-1-A (50 mg, 109.68 μmol) and 2-cyclopropyl-2-hydroxyacetic acid (25.47 mg, 219.36 μmol) of a single configuration were dissolved in N,N-dimethylformamide (2mL), then add HATU (7.57mg, 219.36μmol) and N,N-diisopropylethylamine (42.53mg, 329.04μmol), complete the addition, and react at room temperature for 0.5 hours; use HPLC-MS chromatography Detect the reaction; the reaction solution is directly purified by preparative high performance liquid chromatography to obtain two isomers of the title compound (3-12-A: 12.96 mg, 3-12-B: 13.56 mg).

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

time[min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL/min] 0.00 30 90 28 3.00 30 90 28 18.00 90 10 28

The structural characterization data of 3-12-A (6min LCMS peak is near the front) are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.44 (d, J = 9.0 Hz, 1H), 8.05 (d, J = 10.2 Hz, 1H), 7.33 (s, 1H), 6.54 (s, 1H) ,5.62(q,J＝6.7Hz,1H),5.52(d,J＝5.1Hz,1H),5.42(s,2H),5.24(q,J＝19.2Hz,2H),3.61(dd,J＝ 6.2,5.1Hz,1H),3.32–3.21(m,2H),2.19(q,J＝6.5Hz,2H),1.92–1.80(m,2H),1.26–1.20(m,1H),0.87(t ,J＝7.3Hz,3H),0.57–0.34(m,4H).

ESI-MS(m/z):554.0[M+H]+.

The structural characterization data of 3-12-B (6min LCMS peak appears later) are as follows:

¹ H NMR (400MHz, DMSO-d6) δ8.43 (d, J = 8.7Hz, 1H), 8.06 (d, J = 10.3Hz, 1H), 7.33 (s, 1H), 5.57 (q, J = 6.7 Hz,1H),5.43(s,2H),5.30–5.17(m,2H),3.64(d,J＝6.2Hz,1H),3.29(q,J＝6.7Hz,2H),2.28–2.13(m ,2H),1.93–1.78(m,2H),1.18–1.08(m,1H),0.87(t,J＝7.3Hz,3H),0.50–0.29(m,4H).

ESI-MS(m/z):554.0[M+H]+.

Another single-configuration compound 3-1-B formate (50 mg, 109.68 μmol) and 2-cyclopropyl-2-hydroxyacetic acid (25.47 mg, 219.36 μmol) were dissolved in N,N-dimethyl into formamide (2mL), then add HATU (7.57mg, 219.36μmol) and N,N-diisopropylethylamine (42.53mg, 329.04μmol), complete the addition, and react at room temperature for 0.5 hours; use high performance liquid chromatography mass spectrometry The reaction was detected by chromatography; the reaction solution was directly purified by preparative high-performance liquid chromatography to obtain two isomers of the title compound (3-12-C: 20.19 mg, 3-12-D: 18.33 mg).

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

time[min] Mobile phase A[%] Mobile phase B [%] Flow rate [mL/min] 0.00 30 90 28 3.00 30 90 28 18.00 90 10 28

The structural characterization data of 3-12-C (6min LCMS peak is near the front) are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.47 (d, J = 9.0 Hz, 1H), 8.06 (d, J = 10.3 Hz, 1H), 7.33 (s, 1H), 6.54 (s, 1H) ,5.62(q,J＝6.5Hz,1H),5.53(d,J＝5.1Hz,1H),5.43(s,2H),5.32–5.16(m,2H),3.61(dd,J＝6.3,5.1 Hz,1H),3.32–3.22(m,2H),2.19(q,J＝6.5Hz,2H),1.92–1.80(m,2H),1.28–1.20(m,1H),0.87(t,J＝ 7.3Hz,3H),0.54–0.35(m,4H).

ESI-MS(m/z):554.0[M+H]+.

The structural characterization data of 3-12-D (6min LCMS peak appears later) are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.44(d,J＝8.8Hz,1H),8.05(d,J＝10.2Hz,1H),7.34(s,1H),6.55(s,1H) ,5.58(q,J＝6.7Hz,1H),5.45(d,J＝5.2Hz,1H),5.43(s,2H),5.31–5.14(m,2H),3.65(t,J＝5.7Hz, 1H),3.33–3.21(m,2H),2.28–2.13(m,2H),1.95–1.80(m,2H),1.16–1.09(m,1H),0.88(t,J＝7.3Hz,3H) ,0.46–0.31(m,4H).

ESI-MS(m/z):554.0[M+H] ⁺ .

Example 17: (S)-N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxy-2,3,9,10 ,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)- 2-Hydroxypropylamine and (S)-N-((1R,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxy-2,3,9,10 ,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazine[1,2-b]quinolin-1-yl)-2 -Preparation of hydroxypropylamine

At 25°C, (9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-2,3,12,15-tetrahydrobenzo[de]pyrano[3 ',4':6,7]indolazine[1,2-b]quinoline-10,13(1H,9H)-dione (80.0 mg, 175.5 μmol) and L-lactic acid (31.6 mg, 351.0 μmol ,) was dissolved in DMF (3 mL), then HATU (121.1 mg, 351.0 μmol) and DIPEA (68.0 mg, 526.5 μmol) were added, and the reaction was carried out at room temperature for 2 hr; the reaction solution was directly purified by preparative high-performance liquid chromatography to obtain compound 3-7- A (6.1 mg, yield 12%) and compound 3-7-B (9.6 mg, yield 20%).

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

time[min] Mobile phase A [%] Mobile phase B[%] Flow rate [mL/min] 0.00 30 70 twenty four 2.00 30 70 twenty four 18.00 90 10 twenty four

The structural characterization data of compound 3-7-A (6min LC-MS peak is near the front, retention time is 2.49min):

MS m/z(ESI):528.2[M+H] ⁺

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.55 (d, J = 9.2 Hz, 1H), 8.06 (d, J = 10.4 Hz, 1H), 7.33 (s, 1H), 6.55 (s, 1H) ,6.67(d,J＝4.8Hz,1H),5.65–5.59(m,1H),5.43(s,2H),5.29–5.21(m,1H),5.14–5.10(m,1H),4.15–4.10 (m,,1H),3.27–3.20(m,1H),2.22–2.15(m,2H),1.92–1.81(m,2H),1.41(d,J＝6.8Hz,3H),1.30–1.23( m,1H),0.89–0.85(t,J=7.2Hz,3H).

The structural characterization data of compound 3-7-B (6min LC-MS peak at the rear, retention time 2.50min) are as follows:

MS m/z(ESI):528.2[M+H] ⁺

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.46 (d, J = 8.8 Hz, 1H), 8.06 (d, J = 10.4 Hz, 1H), 7.33 (s, 1H), 6.56 (s, 1H) ,5.60–5.53(ms,1H),5.51(d,J＝5.2Hz,1H),5.43(s,2H),5.27–5.14(m,2H),4.16–4.08(m,1H),3.28–3.22 (m,1H),2.22–2.19(m,2H),1.92–1.81(m,2H),1.49–1.39(m,1H),1.29(d,J＝6.8Hz,3H),0.87(t,J =7.2Hz,3H).

Example 18 N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-1,2,3,9,10,12, 13,15-Octahydrobenzo[de]pyrano[3',4':6,7]indolazine[1,2-b]quinolin-1-yl-1-hydroxycyclopropylcarboxamide Compounds and N-((1R,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-1,2,3,9,10,12,13, 15-Octahydrobenzo[de]pyrano[3',4':6,7]indolazine[1,2-b]quinolin-1-yl-1-hydroxycyclopropylcarboxamide compound synthesis

At 25°C, (9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-2,3,12,15-tetrahydrobenzo[de]pyrano[3 ', 4': 6,7]indolazine[1,2-b]quinoline-10,13(1H,9H)-dione (80mg, 175.49μmol) and 1-hydroxycyclopropanecarboxylic acid (35.83mg ,350.98μmol,) was dissolved in DMF (2mL), then HATU (121.14mg, 350.98μmol) and DIPEA (68.04mg, 526.47μmol) were added. After the addition, react at room temperature for 0.5hr; the reaction solution was concentrated to dryness and directly used with high efficiency The reaction was detected by liquid chromatography-mass spectrometry coupled with chromatography; the reaction solution was directly purified by preparative high-performance liquid chromatography to obtain 5.3 mg of the title compound 3-17-A and 3.5 mg of 3-17-B.

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

time[min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL/min] 0.00 15 85 28 2.00 15 85 28 18.00 90 10 28

The structural characterization data are as follows:

The structural characterization data of 3-17-A (6min LCMS peak is near the front, retention time: 2.657min) are as follows:

ESI-MS(m/z):540.0[M+H]+.

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.69 (d, J = 8.8 Hz, 1H), 8.06 (d, J = 10.2 Hz, 1H), 7.35 (s, 1H), 6.57 (s, 1H) ,6.32(s,1H),5.62(s,1H),5.45(s,2H),5.34–5.24(m,1H),5.20–5.10(m,1H),2.26(s,2H),2.00(s ,1H),1.88(s,2H),1.47–1.12(m,8H),1.01–0.80(m,6H).

The structural characterization data of 3-17-A (6min LCMS peak is later, retention time: 2.724min) are as follows:

ESI-MS(m/z):540.0[M+H]+.

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.69 (d, J = 8.8 Hz, 1H), 8.06 (d, J = 10.2 Hz, 1H), 7.35 (s, 1H), 6.57 (s, 1H) ,6.32(s,1H),5.62(s,1H),5.45(s,2H),5.34–5.24(m,1H),5.20–5.10(m,1H),2.26(s,2H),2.00(s ,1H),1.88(s,2H),1.47–1.12(m,8H),1.01–0.80(m,6H).

Example 19 N-((1S,9S)-4-cyclopropyl-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-1,2,3,9,10,12 ,13,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolazine[1,2-b]quinolin-1-yl)-2-hydroxyacetamide compound Synthesis

Step 1: Synthesis of N-(4-chloro-3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetralin-1-yl)acetamide

Combine N-(4-chloro-3-fluoro-8-oxo-5,6,7,8-tetralin-1-yl)acetamide (570mg, 2.23mmol) and cyclopropylboronic acid (574.56mg, 6.69mmol) was dissolved in 1,4-dioxane, and dichlorodi-tert-butyl-(4-dimethylaminophenyl)phosphine palladium (II) (480mg, 677.97μmol) and cesium carbonate (2.17g , 6.69mmol), after nitrogen protection, react in microwave at 115°C for 2 hours; use high performance liquid chromatography-mass spectrometry to detect the reaction; the reaction solution was diluted with ethyl acetate and filtered, the filtrate was extracted with ethyl acetate (30ml*3), and the organic matter was combined phase, washed with saturated brine (50 mL), dried the organic phase over anhydrous sodium sulfate, and then filtered. The filtrate was evaporated to dryness under reduced pressure. The crude product was purified by column chromatography on a silica gel column (PE: EA = 1:4) to obtain 550 mg of the title compound. .

The structural characterization data are as follows:

ESI-MS(m/z):262.1[M+H] ⁺ .

Step 2: Synthesis of N-(4-cyclopropyl-3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetralin-1-yl)acetamide

Add tetrahydrofuran (30 mL) and tert-butyl alcohol (10 mL) to the reaction solution bottle. After cooling to 5°C in an ice bath, add potassium tert-butoxide (945 mg, 8.42 mmol), and then add N-(4-chloro-3-fluoro -7-(Hydroxyimino)-8-oxo-5,6,7,8-tetralin-1-yl)acetamide (1.0g, 3.83mmol) was dissolved in tetrahydrofuran (1mL) and dropped slowly Add the reaction solution, and then add isoamyl nitrite (718mg, 6.12mmol) after 10 minutes. After the addition is completed, keep the reaction at 5°C for 1 hour; use high performance liquid chromatography mass spectrometry to detect the reaction; use saturated ammonium chloride aqueous solution for the reaction solution. (50mL), extracted with ethyl acetate (40ml*3), combined the organic phases, washed with saturated brine (50mL), dried the organic phase over anhydrous sodium sulfate, then filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain the title compound 1.2g of crude product.

The structural characterization data are as follows:

ESI-MS(m/z):291.1[M+H] ⁺ .

Step 3: Synthesis of N-(7-amino-4-cyclopropyl-3-fluoro-8-oxo-5,6,7,8-tetralin-1-yl)acetamide hydrochloride

The crude product of N-(4-cyclopropyl-3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetralin-1-yl)acetamide (1.2g , 1.41mmol) was dissolved in methanol (7.5mL) and tetrahydrofuran (7.5mL), then 1mol/L hydrochloric acid aqueous solution (7.5mL) and 10% palladium on carbon (450mg) were added. After the addition was completed, the reaction system was replaced three times with a hydrogen balloon. , and reacted at room temperature for 1 hour under a hydrogen atmosphere; the reaction was detected by high-performance liquid chromatography-mass spectrometry coupled with chromatography; the reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain 1.05g of crude product.

The structural characterization data are as follows:

ESI-MS(m/z):277.1[M+H] ⁺ .

Step 4: (9H-fluoren-9-yl)methyl(8-acetamide-5-cyclopropyl-6-fluoro-1-oxo-1,2,3,4-tetralin-2-yl )Synthesis of urethane

The crude hydrochloride of N-(7-amino-4-cyclopropyl-3-fluoro-8-oxo-5,6,7,8-tetralin-1-yl)acetamide (1.05g, 3.80 mmol) in 1,4-dioxane (10 mL), then add sodium bicarbonate (1.3 g, 15.20 mmol), water (10 mL) and 9-fluorenylmethyl-N-succinimidyl carbonate (1.54g, 4.56mmol), after addition, stir and react at room temperature for 2 hours; use high-performance liquid chromatography-mass spectrometry to detect the reaction; pour the reaction solution into water (50mL), and then extract with ethyl acetate (40mL*3) , combine the organic phases, wash with saturated brine (50 mL), dry the organic phase over anhydrous sodium sulfate, filter, and dry the filtrate under reduced pressure to obtain crude product. The crude product was purified by C18 reverse-phase column to obtain 2.0 g of the title compound.

The structural characterization data are as follows:

ESI-MS(m/z):499.2[M+H] ⁺ .

Step 5: (9H-fluoren-9-yl)methyl (8-amino-5-cyclopropyl-6-fluoro-1-oxo-1,2,3,4-tetralin-2-yl) Synthesis of carbamates

(9H-Fluoren-9-yl)methyl(8-acetamide-5-cyclopropyl-6-fluoro-1-oxo-1,2,3,4-tetralin-2-yl)amino Dissolve formate (2.0g, 3.21mmol, 80%) in dioxane (20mL), add 12mol/L concentrated hydrochloric acid (5mL), complete the addition, raise the temperature to 70°C and react for 2 hours; use high-performance liquid phase Mass spectrometry coupled to chromatography was used to detect the reaction; the reaction solution was poured into water (40mL), then extracted with ethyl acetate (30mL*3), the organic phases were combined, washed with saturated brine (40mL), and the organic phase was dried over anhydrous sodium sulfate. Filter, and dry the filtrate under reduced pressure to obtain crude product. The crude product was purified by column chromatography on silica gel (PE:EA=2:1) to obtain 740 mg of the title compound.

The structural characterization data are as follows:

ESI-MS(m/z):457.3[M+H] ⁺ .

Step six: (9H-fluoren-9-yl)methyl ((9S)-4-cyclopropyl-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxyl-2,3, 9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4:6,7]indolazine[1,2-b]quinolin-1-yl)amino Synthesis of formate

(S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolazine-3,6,10(4H)-trione (442 mg ,1.68mmol) and (9H-fluoren-9-yl)methyl (8-amino-5-cyclopropyl-6-fluoro-1-oxo-1,2,3,4-tetralin-2- Base) carbamate (640 mg, 1.40 mmol) was added to toluene (30 mL), and then p-toluenesulfonic acid (242 mg, 1.40 mmol) was added. After the addition was completed, the temperature was raised to 135°C for 2 hours, and the reaction solution was directly heated to 140°C. The crude product was evaporated to dryness under reduced pressure; the crude product was purified by column chromatography on silica gel (DCM: MeOH=33:1) to obtain 1.02g of the title compound.

The structural characterization data are as follows:

ESI-MS(m/z):684.1[M+H] ⁺ .

Step 7: (1S,9S)-1-amino-4-cyclopropyl-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H -Benzo[de]pyrano[3',4':6,7]indolazine[1,2-b]quinoline-10,13-dione (compound 5-29-1)&(1R ,9S)-1-amino-4-cyclopropyl-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de Synthesis of ]pyrano[3',4':6,7]indolazine[1,2-b]quinoline-10,13-dione

(9H-Fluoren-9-yl)methyl((9S)-4-cyclopropyl-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxy-2,3,9, 10,13,15-Hexahydro-1H,12H-benzo[de]pyran[3',4:6,7]indolazine[1,2-b]quinolin-1-yl)carbamic acid Dissolve the ester (1.02g, 1.49mmol) in N,N-dimethylformamide (15mL), then add diethylamine (5ml), complete the addition, and react at room temperature for 0.5 hours; use high-performance liquid chromatography-mass spectrometry to detect Reaction; after the ethylenediamine is evaporated under reduced pressure from the reaction solution, the pH is adjusted to 2-3 with 1 mol/L hydrochloric acid aqueous solution, and the reaction solution is directly purified by preparative high-performance liquid chromatography to obtain two isomers of the title compound (5-22 -7-A: 60mg; 5-22-7-B: 55mg).

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

time[min] Mobile phase A[%] Mobile phase B [%] Flow rate [mL/min] 0.00 10 90 28 3.00 10 90 28 18.00 70 30 28

The structural characterization data of 5-22-7-A (6min LCMS peak is near the front, retention time: 2.28min) are as follows:

ESI-MS(m/z):462.2[M+H] ⁺ .

The structural characterization data of 5-22-7-B (6min LCMS peak is later, retention time: 2.35min) are as follows:

ESI-MS(m/z):462.2[M+H] ⁺ .

Step 8 N-((1S,9S)-4-cyclopropyl-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-1,2,3,9,10,12,13 ,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolazine[1,2-b]quinolin-1-yl)-2-hydroxyacetamide and N- ((1R,9S)-4-cyclopropyl-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-1,2,3,9,10,12,13,15-tetra Synthesis of Hydrobenzo[de]pyrano[3',4':6,7]indolazine[1,2-b]quinolin-1-yl)-2-hydroxyacetamide

At 25°C, single-configuration compound 5-28-7-A (40 mg, 86 μmol) and glycolic acid (8 mg, 104 μmol,) were dissolved in DMF (2 mL), and then HATU (40 mg, 104 μmol) and DIPEA ( 36 mg, 258 μmol), after addition, react at room temperature for 0.5 hr; the reaction solution was concentrated to dryness and directly detected by HPLC-MS/MS; the reaction solution was directly purified by preparative HPLC to obtain 12.5 mg of compound 5-22-A. .

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

time[min] Mobile phase A[%] Mobile phase B [%] Flow rate [mL/min] 0.00 10 90 28 2.00 10 90 28 18.00 90 10 28

The structural characterization data are as follows:

The structural characterization data of 5-22-A (6min LCMS peak is near the front, retention time: 2.540min) are as follows:

ESI-MS(m/z):520.0[M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.46 (d, J = 8.9 Hz, 1H), 7.74 (d, J = 11.9 Hz, 1H), 7.30 (s, 1H), 6.53 (s, 1H) ,5.64–5.56(m,1H),5.49(t,J＝5.8Hz,1H),5.42(s,2H),5.19(s,2H),3.96(d,J＝5.7Hz,2H),2.25– 2.10(m,2H),2.04–1.79(m,4H),1.23(s,2H),1.15–1.05(m,2H),0.87(t,J＝7.2Hz,3H),0.80–0.70(m, 2H).

At 25°C, single-configuration compound 5-28-7-B (30 mg, 65 μmol) and glycolic acid (6 mg, 78 μmol) were dissolved in DMF (2 mL), and then HATU (40 mg, 104 μmol) and DIPEA ( 17 mg, 130 μmol), after addition, react at room temperature for 0.5 hr; the reaction solution is concentrated to dryness and directly detected by HPLC-MS/MS; the reaction solution is directly purified by preparative HPLC to obtain 13.83 mg of compound 5-22-B. .

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

time[min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL/min] 0.00 10 90 28 2.00 10 90 28 18.00 90 10 28

The structural characterization data are as follows:

The structural characterization data of 5-22-B (6min LCMS peak is near the front, retention time: 2.612min) are as follows:

ESI-MS(m/z):520.0[M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.49 (d, J = 8.9 Hz, 1H), 7.74 (d, J = 11.9 Hz, 1H), 7.31 (s, 1H), 6.53 (s, 1H) ,5.60(s,1H),5.51(t,J＝5.9Hz,1H),5.43(s,2H),5.25–5.13(m,2H),3.97(d,J＝5.8Hz,2H),2.18( s,2H),2.04–1.91(m,4H),1.90–1.80(m,1H),1.23(s,6H),1.15–1.05(m,2H),0.87(t,J＝7.2Hz,4H) ,0.80–0.70(m,2H).

Example 20 (R)-3-(dimethylamine)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo -1,2,3,9,10,12,13,15-Octahydrobenzo[de]pyrano[3',4':6,7]indolazine[1,2-b]quinoline -1-yl)-2-hydroxypropionamide and (S)-3-(dimethylamine)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl -10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazine[ Synthesis of 1,2-b]quinolin-1-yl)-2-hydroxypropionamide

Step 1: (9H-Fluoro-9-yl)methyl((S)-3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13 -Dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazine[1,2-b ]quinolin-1-yl)amino)-2-hydroxy-3-oxypropyl)carbamate and (9H-fluoro-9-yl)methyl((R)-3-(((1S,9S )-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de] Synthesis of pyrano[3',4':6,7]indolazine[1,2-b]quinolin-1-yl)amino)-2-hydroxy-3-oxypropyl)carbamate

At 25°C, (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3,12,15-benzo[de]pyrano[ 3',4':6,7]indolazine[1,2-b]quinoline-10,13(1H,9H)-dionemethanesulfonate (72mg, 166μmol) and 3-(((( 9H-Fluoren-9-yl)methoxy)carbonyl)amino)-2-hydroxypropionic acid (65 mg, 199 μmol,) was dissolved in DMF (2 mL), and then HATU (95 mg, 250 μmol) and DIPEA (65 mg, 498 μmol) were added ), after addition, react at room temperature for 0.5 hr; the reaction solution is concentrated to dryness and directly detected by HPLC-MS/MS; the reaction solution is directly purified by preparative HPLC to obtain the title compound (compound 1-10-1-A ) 24 mg and (compound 1-10-1-B) 28 mg.

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

time[min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL/min] 0.00 15 85 28 2.00 15 85 28 18.00 90 10 28

The structural characterization data of compound 1-10-1-A (6min LCMS peak is near the front, retention time: 3.283min) are as follows:

ESI-MS(m/z):745.4[M+H] ⁺ .

The structural characterization data of compound 1-10-1-B (6min LCMS peak is late, retention time: 3.465min) are as follows:

ESI-MS(m/z):745.4[M+H] ⁺ .

Step 2: (R)-3-amino-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2, 3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazine[1,2-b]quinolin-1-yl) -Synthesis of 2-hydroxypropionamide and (S)-3-amino-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy Generation-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazine[1,2-b]quino Synthesis of pholin-1-yl)-2-hydroxypropionamide

Dissolve compound 1-10-1-B (28 mg, 37 μmol) in DMF (2 mL) at 25°C, then add diethylamine (1 mL). After the addition, react at room temperature for 1.0 hr; the reaction solution is concentrated to dryness to obtain The crude product (compound 1-10-2-B), 28 mg, was directly used in the next reaction.

Dissolve compound 1-10-1-A (24 mg, 33 μmol) in DMF (2 mL) at 25°C, then add diethylamine (1 mL). After the addition, react at room temperature for 1.0 hr; the reaction solution is concentrated to dryness to obtain The crude product (compound 1-10-2-A), 24 mg, was directly used in the next reaction.

The structural characterization data are as follows:

ESI-MS(m/z):523.2[M+H] ⁺ .

Step 3: (R)-3-(dimethylamine)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo- 1,2,3,9,10,12,13,15-Octahydrobenzo[de]pyrano[3',4':6,7]indolazine[1,2-b]quinoline- Synthesis of 1-yl)-2-hydroxypropionamide and (S)-3-(dimethylamine)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl Base-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazine Synthesis of [1,2-b]quinolin-1-yl)-2-hydroxypropionamide

Dissolve compound 1-10-2-B (28 mg, 37 μmol, 70%) in methanol (2 mL) at 25°C, then add formaldehyde aqueous solution (1 mL). After the addition, react at room temperature for 16.0 hr, then add Sodium cyanoborohydride (7.07 mg, 96.45 μmol), reacted at room temperature for 1.0 hr; the reaction solution was concentrated to dryness and directly used high performance liquid chromatography mass spectrometry to detect the reaction; the reaction solution was directly purified by preparative high performance liquid chromatography to obtain the title compound (compound 1-10B)1.3mg.

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: methanol; mobile phase B: water (0.05% formic acid)

time[min] Mobile phase A[%] Mobile phase B [%] Flow rate [mL/min] 0.00 15 85 28 2.00 15 85 28 18.00 90 10 28

The structural characterization data of compound 1-10-B (6min LCMS peak is near the front, retention time: 1.937min) are as follows:

ESI-MS(m/z):551.2[M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.48 (d, J = 8.6 Hz, 1H), 8.31 (s, 2H), 7.80 (d, J = 11.0 Hz, 1H), 7.31 (s, 1H) ,6.55(s,1H),5.54(s,1H),5.43(s,2H),5.34(d,J＝19.2Hz,1H),5.19(d,J＝19.1Hz,1H),4.09–4.06( m,1H),3.20–3.15(m,2H),2.59–2.53(m,1H),2.45–2.42(m,1H),2.42–2.38(s,3H),2.23–2.19(d,J＝7.0 Hz,1H),2.13(s,6H),2.12–2.08(m,1H),2.02 –1.95(m,1H),1.90–1.85(m,2H),1.23(s,2H),0.88(d, J＝7.2Hz,3H).

Dissolve compound 1-10-2-A (24 mg, 33 μmol, 70%) in methanol (2 mL) at 25°C, then add formaldehyde aqueous solution (1 mL). After the addition, react at room temperature for 16.0 hr, then add Sodium cyanoborohydride (6.06 mg, 96.45 μmol), reacted at room temperature for 1.0 hr; the reaction solution was concentrated to dryness and directly used high performance liquid chromatography mass spectrometry to detect the reaction; the reaction solution was directly purified by preparative high performance liquid chromatography to obtain the title compound (compound 1-10-A)4.44mg.

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: methanol; mobile phase B: water (0.05% formic acid)

time[min] Mobile phase A [%] Mobile phase B[%] Flow rate [mL/min] 0.00 15 85 28 2.00 15 85 28 18.00 90 10 28

The structural characterization data of compound 1-10-A (6min LCMS peak is near the front, retention time: 1.920min) are as follows:

ESI-MS(m/z):551.2[M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.58 (d, J = 9.0 Hz, 1H), 8.28 (s, 1H), 7.84 (d, J = 10.9 Hz, 1H), 7.37 (s, 1H) ,6.61(s,1H),5.66–5.59(m,1H),5.49(s,2H),5.35(d,J＝19.1Hz,1H),5.17(d,J＝18.9Hz,1H),4.23– 4.16(m,1H),3.23(d,J＝7.8Hz,2H),2.75–2.67(m,2H),2.45(s,3H),2.30(s,6H),2.27–2.17(m,2H) ,2.14–1.99(m,1H),1.98–1.87(m,2H),1.30(s,2H),0.93(t,J=7.3Hz,3H).

Example 21 (S)-14-(2-(cyclopropylamino)ethyl)-7-ethyl-7-hydroxy-7H-[1,3]dioxola[4,5- g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(10H,13H)-dione (compound 4-14)

Step 1: Synthesis of 2-nitro-4,5-methylenedioxyacetophenone

Compound 4-14-1 (10.0g, 60.92mmol) was dissolved in nitromethane (100mL), concentrated nitric acid (26mL) was slowly added dropwise while stirring, and the reaction was carried out at room temperature for 2 hours. TLC monitors the reaction. A small amount of raw material remains and the product is obvious. Slowly drop in saturated sodium bicarbonate aqueous solution to neutralize the reaction, add methylene chloride for extraction three times, combine the organic phases, wash with saturated brine three times, dry, and concentrate to obtain a crude product. Silica gel column purification (eluent: 0-20% ethyl acetate/petroleum ether) gave 9.8 g of the title compound.

Step 2: Synthesis of 6-amino-3,4-methylenedioxyacetophenone

Compound 4-14-2 (2.0g, 9.56mmol) was dissolved in ethyl acetate (20mL), 10% palladium on carbon (0.2g) was added, hydrogen was substituted, and the reaction was stirred for 4 hours under protection. After filtration, the filtrate was concentrated under reduced pressure to obtain 1.7 g of crude product of the title compound.

Step 3: Synthesis of 6-acetamido-3,4-methylenedioxyacetophenone

Compound 4-14-3 (1.7g, 9.49mmol) was dissolved in acetic anhydride (17mL), and the reaction was stirred for 1 hour. The solvent was evaporated under reduced pressure, water was added, stirred, and filtered. The solid was washed with water and dried under vacuum to obtain 2.08 g of crude title compound.

Step 4: Synthesis of (E)-N-(6-(3-(dimethylamino)acryloyl)benzo[d][1,3]dioxol-5-yl)acetamide

Compound 4-14-4 (1.88g, 8.50mmol) was dissolved in DMF-DMA (30mL), and the temperature was raised to 120°C for 2 hours. After the solvent was evaporated under reduced pressure, 2.33 g of crude title compound was obtained.

The structural characterization data are as follows:

ESI-MS(m/z):277.2[M+1] ⁺ .

Step 5: Synthesis of (E)-N-(6-(3-(cyclopropylamino)acryloyl)benzo[d][1,3]dioxol-5-yl)acetamide

Compound 4-14-5 (200 mg, 0.72 mmol) was dissolved in ethanol (5 mL), cyclopropylamine (413.3 mg, 7.24 mmol) was added dropwise, and the temperature was raised to 50°C for 16 hours. The solvent was evaporated under reduced pressure to obtain 208 mg of crude title compound.

The structural characterization data are as follows:

ESI-MS(m/z):289.2[M+1] ⁺ .

Step 6: Synthesis of N-(6-(3-(cyclopropylamino)propionyl)benzo[d][1,3]dioxol-5-yl)acetamide

Compound 4-14-6 (208 mg, 0.72 mmol) was dissolved in glacial acetic acid (4 mL). Sodium borohydride (13.65 mg, 0.36 mmol) was added under cooling and stirring in an ice-water bath, and the solution was returned to room temperature and stirred for 3 hours. The solvent was evaporated under reduced pressure to obtain 209 mg of crude title compound.

The structural characterization data are as follows:

ESI-MS(m/z):291.1[M+1] ⁺ .

Step 7: (9H-Fluoren-9-yl)methyl(3-(6-acetylaminobenzo[d][1,3]dioxol-5-yl)-3-oxypropyl)( Synthesis of cyclopropyl)carbamate

Compound 4-14-7 (200 mg, 0.69 mmol) was dissolved in 1,4-dioxane (20 mL) and water (20 mL), and 9-fluorenylmethyl-N-succinimidyl carbonate was added under stirring. (395 mg, 0.68 mmol) and sodium bicarbonate (231.5 mg, 2.76 mmol), react at room temperature for 2 hours. Add water and ethyl acetate, stir, let stand for liquid separation, wash the organic phase with saturated brine, dry and concentrate, and purify on silica gel column (eluent: 30% ethyl acetate/petroleum ether) to obtain 350 mg of the title compound.

Step 8: (9H-fluoren-9-yl)methyl (3-(6-aminobenzo[d][1,3]dioxol-5-yl)-3-oxopropyl) (cyclo Synthesis of propyl) carbamate

Compound 4-14-8 (350 mg, 0.68 mmol) was dissolved in 1,4-dioxane (10 mL), 3N hydrochloric acid aqueous solution (10 mL) was added dropwise, and the temperature was raised to 60°C and the reaction was stirred for 16 hours. Add water and ethyl acetate, stir, let stand for liquid separation, wash the organic phase with water, dry and concentrate, and purify on silica gel column (eluent: 33% ethyl acetate/petroleum ether) to obtain 218 mg of the title compound.

Step 9: (S)-(9H-fluoren-9-yl)methylcyclopropyl(2-(7-ethyl-7-hydroxy-8,11-dioxy-8,10,11,13- Tetrahydro-7H-[1,3]dioxola[4,5-g]pyrano[3,4]:6,7]indolizino[1,2-b]quinoline- Synthesis of 14-yl)ethyl)carbamate

Compound 4-14-9 (40 mg, 0.085 mmol) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyran[3,4-f]indolazine-3 , 6,10(4H)-trione (24.62mg, 0.094mmol) was dissolved in toluene (1mL), p-toluenesulfonic acid (2.93mg, 0.017mmol) was added, and the temperature was raised to 120°C for 4 hours. Concentrate under reduced pressure to obtain 59 mg of crude title compound.

The structural characterization data are as follows:

ESI-MS(m/z):698.1[M+1] ⁺ .

Step 10: (S)-14-(2-(cyclopropylamino)ethyl)-7-ethyl-7-hydroxy-7H-[1,3]dioxola[4,5-g] Synthesis of pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(10H,13H)-dione

Compound 4-14-10 (59 mg, 0.085 mmol) was dissolved in DMF (1 mL), diethylamine (0.5 mL) was added dropwise, and the reaction was stirred for 1 hour. Diethylamine was evaporated under reduced pressure, and 3N hydrochloric acid was added dropwise to acidify the solution. It was preparatively purified by high-performance liquid phase (purification conditions are as follows), and lyophilized to obtain 12.66 mg of the trifluoroacetate salt of the title compound.

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% trifluoroacetic acid)

time[min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL/min] 0 20 80 28 2 20 80 28 18 80 20 28

The structural characterization data are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.73(s,2H),7.67(s,1H),7.57(s,1H),7.26(s,1H),6.54(s,1H),6.33( s,2H),5.44(s,2H),5.34(s,2H),3.40(s,4H),2.82(s,1H),1.91-1.81(m,2H),0.87(t,J＝7.2Hz ,5H),0.79(d,J=7.4Hz,2H).

ESI-MS(m/z):476.1[M+1] ⁺ .

Example 22 (S)-7-ethyl-7-hydroxy-14-(2-((2-methoxyethyl)amino)ethyl)-7H-[1,3]dioxetane And[4,5-g]pyrano[3',4':6,7]indolazine[1,2-b]quinoline-8,11(10H,13H)-dione (compound 4- 15)

Step 1: (E)-N-(6-(3-((2-methoxyethyl)amino)acryloyl)benzo[d][1,3]dioxetane-5-yl)ethyl Synthesis of amides

Compound 4-14-5 (200 mg, 0.72 mmol) was dissolved in ethanol (5 mL), 2-methoxyethylamine (543.7 mg, 7.24 mmol) was added dropwise, and the temperature was raised to 50°C for 16 hours. LCMS monitors the reaction. There is a small amount of raw material remaining and the product is obvious. The solvent was evaporated under reduced pressure to obtain 221 mg of the title compound, which was directly used for the next reaction.

The structural characterization data are as follows:

ESI-MS(m/z):307.1[M+1] ⁺

Step 2: Synthesis of N-(6-(3-((2-methoxyethyl)amino)propionyl)benzo[d][1,3]dioxetane-5-yl)acetamide

Compound 4-15-1 (200 mg, 0.65 mmol) was dissolved in glacial acetic acid (4 mL). Sodium borohydride (12.35 mg, 0.33 mmol) was added under cooling and stirring in an ice-water bath, and the mixture was returned to room temperature and stirred for 3 hours. The reaction was monitored by LCMS. The starting material disappeared and the product was obvious. The solvent was evaporated under reduced pressure to obtain 200 mg of the title compound, which was directly used for the next reaction.

ESI-MS(m/z):309.1[M+1] ⁺

Step 3: (9H-fluoren-9-yl)methyl(3-(6-acetylaminobenzo[d][1,3]dioxol-5-yl)-3-oxypropyl)(2- Synthesis of Methoxyethyl) Carbamate

The crude product of compound 4-15-2 (200 mg, 0.65 mmol) was dissolved in 1,4-dioxane (20 mL) and water (20 mL), and 9-fluorenylmethyl-N-succinimidyl was added under stirring. Carbonate (372 mg, 0.65 mmol) and sodium bicarbonate (231.5 mg, 2.76 mmol) were reacted at room temperature for 2 hours. TLC monitors that the raw materials disappear and the product is obvious. Add water and ethyl acetate, stir, let stand for liquid separation, wash the organic phase with saturated brine, dry and concentrate, and purify on a silica gel column (eluent: 50% ethyl acetate/petroleum ether) to obtain 180 mg of the title compound.

Step 4: ((9H-fluoren-9-yl)methyl(3-(6-aminobenzo[d][1,3]dioxin-5-yl)-3-oxypropyl)(2- Synthesis of Methoxyethyl) Carbamate

Compound 4-15-3 (180 mg, 0.68 mmol) was dissolved in 1,4-dioxane (5 mL), 3N hydrochloric acid aqueous solution (5 mL) was added dropwise, and the temperature was raised to 60°C and the reaction was stirred for 16 hours. Add water and ethyl acetate, stir, let stand for liquid separation, wash the organic phase with water, dry and concentrate, and purify on silica gel column (eluent: 45% ethyl acetate/petroleum ether) to obtain 132 mg of the title compound.

Step 5: (S)-(9H-fluoren-9-yl)methyl(2-(7-ethyl-7-hydroxy-8,11-dioxy-8,10,11,13-tetrahydro- 7H-[1,3]dioxacyclo[4,5-g]pyrano[3',4]:6,7]indolizino[1,2-b]quinolin-14-yl Synthesis of )ethyl)(2-methoxyethyl)carbamate

Compound 4-15-4 (130 mg, 0.266 mmol) and rac-(4S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indole Azine-3,6,10-trione (70.05 mg, 0.266 mmol) was dissolved in toluene (4 mL), p-toluenesulfonic acid (9.16 mg, 0.053 mmol) was added, and the temperature was raised to 120°C for 4 hours. The reaction was monitored by LCMS. The starting material disappeared and the product was obvious. Concentrate under reduced pressure to obtain 190 mg of crude product of the title compound.

The structural characterization data are as follows:

ESI-MS(m/z):716.1[M+1] ⁺

Step six: (S)-7-ethyl-7-hydroxy-14-(2-((2-methoxyethyl)amino)ethyl)-7H-[1,3]dioxona[ Synthesis of 4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(10H,13H)-dione

Compound 4-15-5 (190 mg, 0.265 mmol) was dissolved in DMF (3 mL), diethylamine (2 mL) was added dropwise, and the reaction was stirred for 1 hour. The reaction was monitored by LCMS. The starting material disappeared and the product was obvious. Diethylamine was evaporated under reduced pressure, and 3N hydrochloric acid was added dropwise for acidification, followed by purification and freeze-drying to obtain 99.28 mg of the title compound.

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

time[min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL/min] 0 20 80 28 2 20 80 28 18 80 20 28

The structural characterization data are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.68(s,2H),7.68(s,1H),7.56(s,1H),7.26(s,1H),6.52(s,1H),6.32 (s,2H),5.44(s,2H),5.31(s,2H),3.65–3.58(m,2H),3.42(d,J＝10.2Hz, 2H),3.36(s,3H),3.22( d,J＝4.0Hz,4H),1.94–1.80(m,2H),0.87(t,J＝7.3Hz,3H).

ESI-MS(m/z):494.2[M+1] ⁺

Example 23 N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-10,13-dioxy-2,3,9,10,13 ,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolazine[1,2-b]quinolin-1-yl)-2-hydroxyethyl Amide&N-((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-10,13-dioxy-2,3,9,10,13,15-six Hydrogen-1H,12H-benzo[de]pyrano[3',4:6,7]indolazine[1,2-b]quinolin-1-yl)-2-hydroxyacetamide

Step 1: Synthesis of N-(3-bromo-5-fluoro-4-methoxyphenyl)acetamide

Dissolve 3-bromo-5-fluoro-4-methoxy-aniline (1.7g, 7.73mmol) in tetrahydrofuran (30mL), then add triethylamine (2.35g, 23.18mmol) and acetic anhydride (1.18g, 11.59mmol), after adding, raise the temperature to 50°C and stir for 4 hours; use high performance liquid chromatography-mass spectrometry to detect the reaction; cool the reaction solution to room temperature, dilute it with ethyl acetate (50mL), and then use clear water (30mL) and saturated brine (30 mL) were washed once each, and the organic phase was separated, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain a crude product, which was purified by beating with (petroleum ether: ethyl acetate = 5:1) to obtain the title compound. 1.1g.

The structural characterization data are as follows:

ESI-MS(m/z):262.0[M+H] ⁺ .

Step 2: Synthesis of (E)-4-(5-acetylamino-3-fluoro-2-methoxyphenyl)-3-butenoic acid

Dissolve N-(3-bromo-5-fluoro-4-methoxyphenyl)acetamide (1.1g, 4.20mmol) and 3-butenoic acid (397.47mg, 4.62mmol) in 1,4 dioxane To a mixed solvent of ring (20mL) and water (5mL), triethylamine (1.27g, 12.59mmol), tris(o-methylphenyl)phosphorus (127.75mg, 419.73μmol) and palladium acetate (47.12mg) were added. 209.89 μmol), after addition, the reaction system was replaced with nitrogen three times, and the temperature was raised to 100°C for 4 hours in a nitrogen atmosphere; the reaction was detected by high performance liquid chromatography-mass spectrometry coupled with chromatography; after the reaction solution was cooled to room temperature, 1 mol/L Aqueous sodium hydroxide solution (50 mL) and ethyl acetate (50 mL) were separated by shaking. After separating the lower aqueous phase, adjust the pH to about 3 with 4mol/L hydrochloric acid aqueous solution, then extract with ethyl acetate (40mL*2), wash the combined organic phases with saturated brine (40mL), dry over anhydrous sodium sulfate, and filter , the filtrate was evaporated to dryness under reduced pressure to obtain 1.1 g of crude product of the title compound.

The structural characterization data are as follows:

ESI-MS(m/z):268.1[M+H] ⁺ .

Step 3: Synthesis of 4-(5-acetamido-3-fluoro-2-methoxyphenyl)butyric acid

Dissolve the crude product of (E)-4-(5-acetamido-3-fluoro-2-methoxyphenyl)-3-butenoic acid (1.1g, 4.12mmol,) in methanol (20mL), and then add 10% palladium on carbon (100 mg) was added, and then the reaction system was replaced three times with a hydrogen balloon, and reacted under a hydrogen atmosphere for 4 hours; the reaction was detected by high performance liquid chromatography mass spectrometry coupled with chromatography; the reaction liquid was filtered, and the filtrate was concentrated to dryness under reduced pressure. The crude product of the title compound was 1.05 g.

The structural characterization data are as follows:

ESI-MS(m/z):270.1[M+H] ⁺ .

Step 4: Synthesis of N-(3-fluoro-4-methoxy-8-oxy-5,6,7,8-tetralin-1-yl)acetamide

Dissolve the crude product of 4-(5-acetamido-3-fluoro-2-methoxyphenyl)butyric acid (1.1g, 4.09mmol) in trifluoroacetic acid (10mL), cool to 5°C, and slowly add After adding trifluoroacetic anhydride (4.29g, 20.43mmol), let it naturally rise to room temperature and react for 2 hours; use high performance liquid chromatography-mass spectrometry to detect the reaction; slowly pour the reaction solution into water (60mL), and then use ethyl acetate (40mL*3) extraction, combine the organic phases, wash with saturated sodium bicarbonate aqueous solution until neutral, then wash with saturated brine (40mL), dry the organic phase with anhydrous sodium sulfate, then filter, and the filtrate is evaporated to dryness under reduced pressure to obtain The crude product was purified through a flash silica gel column (ethyl acetate: petroleum ether = 0-40%) to obtain 503 mg of the title compound.

The structural characterization data are as follows:

ESI-MS(m/z):252.1[M+H] ⁺ .

Step 5: Synthesis of N-(3-fluoro-7-(hydroxyimino)-4-methoxy-8-oxy-5,6,7,8-tetralin-1-yl)acetamide

Add tetrahydrofuran (15 mL) and tert-butyl alcohol (4 mL) to the reaction solution bottle. After cooling to 5°C in an ice bath, add potassium tert-butoxide (491.26 mg, 4.38 mmol), and then add N-(3-fluoro-4- Methoxy-8-oxy-5,6,7,8-tetralin-1-yl)acetamide (500 mg, 1.99 mmol) was dissolved in tetrahydrofuran (5 mL), and slowly added dropwise to the reaction solution for 10 minutes Then add isoamyl nitrite (373.01 mg, 3.18 mmol), complete the addition, and maintain the reaction at 5°C for 1 hour; use high-performance liquid chromatography-mass spectrometry to detect the reaction; the reaction solution is quenched with saturated ammonium chloride aqueous solution (50 mL) Then, extract with ethyl acetate (40*2), combine the organic phases, wash with saturated brine (40 mL), dry the organic phase with anhydrous sodium sulfate, then filter, and evaporate the filtrate to dryness under reduced pressure to obtain 550 mg of the crude product of the title compound.

The structural characterization data are as follows:

ESI-MS(m/z):281.1[M+H] ⁺ .

Step 6: Synthesis of N-(7-amino-3-fluoro-4-methoxy-8-oxy-5,6,7,8-tetralin-1-yl)acetamide

The crude product of N-(3-fluoro-7-(hydroxyimino)-4-methoxy-8-oxy-5,6,7,8-tetralin-1-yl)acetamide (520 mg, 1.86 mmol) was dissolved in a mixed solution of methanol (10 mL) and tetrahydrofuran (10 mL), and then 1 mol/L hydrochloric acid aqueous solution (3.71 mL) and 10% palladium on carbon (50 mg) were added. After the addition was completed, the reaction system was replaced three times with a hydrogen balloon. , and reacted at room temperature for 1 hour under a hydrogen atmosphere; the reaction was detected by high-performance liquid chromatography-mass spectrometry coupled with chromatography; the reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain 551 mg of crude hydrochloride of the title compound.

The structural characterization data are as follows:

ESI-MS(m/z):267.1[M+H] ⁺ .

Step 7: (9H-fluoren-9-yl)methyl(8-acetamide-6-fluoro-5-methoxy-1-oxy-1,2,3,4-tetralin-2-yl )Synthesis of urethane

The crude hydrochloride of N-(7-amino-3-fluoro-4-methoxy-8-oxy-5,6,7,8-tetralin-1-yl)acetamide (550 mg, 1.64 mmol) in 1,4-dioxane (15 mL), then sodium bicarbonate (549.45 mg, 6.54 mmol), water (5 mL) and 9-fluorenylmethyl-N-succinimidyl carbonate were added (1.12g, 1.96mmol), after addition, stir and react at room temperature for 2 hours; use high performance liquid chromatography-mass spectrometry to detect the reaction; pour the reaction solution into water (50mL), and then extract with ethyl acetate (40mL*2) , combine the organic phases, wash with saturated brine (40 mL), dry the organic phase over anhydrous sodium sulfate, filter, and dry the filtrate under reduced pressure to obtain crude product. The crude product was purified by C18 (acetonitrile/0.05% formic acid aqueous solution, 20% acetonitrile to 100% acetonitrile) reverse phase column to obtain 410 mg of the title compound.

The structural characterization data are as follows:

ESI-MS(m/z):489.1[M+H] ⁺ .

Step 8: (9H-fluoren-9-yl)methyl (8-amino-6-fluoro-5-methoxy-1-oxy-1,2,3,4-tetralin-2-yl) Synthesis of carbamates

(9H-Fluoren-9-yl)methyl(8-acetamide-6-fluoro-5-methoxy-1-oxy-1,2,3,4-tetralin-2-yl)amino Dissolve formate (410 mg, 839.29 μmol) in dioxane (10 mL), add 12 mol/L concentrated hydrochloric acid (2 mL), complete the addition, raise the temperature to 70°C and react for 2 hours; use HPLC-MS chromatography Detect the reaction; pour the reaction solution into water (30mL), then extract with ethyl acetate (30mL*2), combine the organic phases, wash with saturated brine (30mL), dry the organic phase over anhydrous sodium sulfate, filter, and reduce the filtrate Press and dry to get a rough product. Purified via flash silica gel (ethyl acetate: petroleum ether = 0-60%) to obtain 351 mg of the title compound.

The structural characterization data are as follows:

ESI-MS(m/z):447.1[M+H] ⁺ .

Step 9: (9H-fluoren-9-yl)methyl ((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-10,13-dioxy-2,3, 9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4:6,7]indolazine[1,2-b]quinolin-1-yl)amino Synthesis of formate

(S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolazine-3,6,10(4H)-trione (247.64 mg, 940.71 μmol) and (9H-fluoren-9-yl)methyl (8-amino-6-fluoro-5-methoxy-1-oxy-1,2,3,4-tetralin-2 -Based) carbamate (350 mg, 783.93 μmol) was added to toluene (15 mL), and then p-toluenesulfonic acid (134.84 mg, 783.93 μmol) was added. After the addition was completed, the temperature was raised to 135°C and reacted for 4 hours. The reaction solution was directly The crude product was evaporated to dryness under reduced pressure at 135°C; the crude product was purified by flash silica gel (methanol:dichloromethane=0-6%) column to obtain 358 mg of the title compound.

The structural characterization data are as follows:

ESI-MS(m/z):674.2[M+H] ⁺ .

Step 10: (9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-1,2,3,9,12,15-hexahydro-10H,13H-benzene Synthesis of [de]pyrano[3',4':6,7]indolazine[1,2-b]quinoline-10,13-dione

(9H-Fluoren-9-yl)methyl((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-10,13-dioxy-2,3,9, 10,13,15-Hexahydro-1H,12H-benzo[de]pyran[3',4:6,7]indolazine[1,2-b]quinolin-1-yl)carbamic acid The ester (358 mg, 531.41 μmol) was dissolved in N,N-dimethylformamide (4 mL), then diethylamine (0.4 mL) was added, the addition was completed, and the reaction was carried out at room temperature for 0.5 hours; detected by high-performance liquid chromatography-mass spectrometry. Reaction; the reaction solution was evaporated to dryness under reduced pressure to obtain a crude product, which was purified by beating with ethyl acetate to obtain 220 mg of the title compound.

The structural characterization data are as follows:

ESI-MS(m/z):452.1[M+H] ⁺ .

Step 11: N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-10,13-dioxy-2,3,9,10,13, 15-Hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolazine[1,2-b]quinolin-1-yl)-2-hydroxyacetamide &N-((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-10,13-dioxy-2,3,9,10,13,15-hexahydro Synthesis of -1H,12H-benzo[de]pyrano[3',4:6,7]indolazine[1,2-b]quinolin-1-yl)-2-hydroxyacetamide

(9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[ de]pyrano[3',4':6,7]indolazine[1,2-b]quinoline-10,13-dione (50 mg, 110.76 μmol) and 2-glycolic acid (16.85 mg, 221.51μmol) was dissolved in N,N-dimethylformamide (2mL), then HATU (84.17mg, 221.51μmol) and N,N-diisopropylethylamine (42.94mg, 332.27μmol) were added. After completion, react at room temperature for 0.5 hours; use high-performance liquid chromatography-mass spectrometry to detect the reaction; the reaction solution is directly purified by preparative high-performance liquid chromatography to obtain two isomers of the title compound with a single configuration (5-34-A: 6.22 mg, 5-34-B: 9.81mg)

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

time[min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL/min] 0.00 10 90 28 2.00 10 90 28 18.00 90 10 28

The structural characterization data of 5-34-A are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.53 (d, J = 9.0Hz, 1H), 7.89 (d, J = 12.3Hz, 1H), 7.33 (s, 1H), 5.58 (q, J = 7.5,7.0Hz,1H),5.42(d,J＝2.1Hz,2H),5.25–5.09(m,2H),3.98(s,2H),3.96(d,J＝1.1Hz,3H),3.30– 3.10(m,2H),2.15(q,J＝7.4Hz,2H),1.93–1.80(m,2H),0.87(t,J＝7.3Hz,3H).

ESI-MS(m/z):510.2[M+H] ⁺ .

The structural characterization data of 5-34-B are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.55 (d, J = 9.0Hz, 1H), 7.90 (d, J = 12.3Hz, 1H), 7.34 (s, 1H), 5.59 (q, J = 7.4,6.8Hz,1H),5.43(s,2H),5.25–5.11(m,2H),3.99(s,2H),3.96 (d,J＝1.1Hz,3H),3.30–3.13(m,2H ),2.15(q,J＝6.4Hz,2H),1.93–1.82(m,2H),0.88(t,J＝7.3Hz,3H).

ESI-MS(m/z):510.2[M+H] ⁺ .

Example 24 (2R)-N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxy-2,3,9, 10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolazine[1,2-b]quinolin-1-yl)-2 -Hydroxypropylamine&(2R)-N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10 ,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolazine[1,2-b]quinolin-1-yl)-2- Hydroxypropylamine

Step 1: (2R)-N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10, 13,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolazine[1,2-b]quinolin-1-yl)-2-hydroxy Propylamine&(2R)-N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13 ,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolazine[1,2-b]quinolin-1-yl)-2-hydroxypropylamine Synthesis

(9S)-1-Amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de ]pyrano[3',4':6,7]indolazine[1,2-b]quinoline-10,13-dione (50 mg, 110.64 μmol) and (2R)-2-hydroxypropionic acid (19.93mg, 221.29μmol) was dissolved in N,N-dimethylformamide (2mL), then HATU (84.09mg, 221.29μmol) and N,N-diisopropylethylamine (42.90mg, 331.93 μmol), after addition, react at room temperature for 0.5 hours; use high-performance liquid chromatography-mass spectrometry to detect the reaction; the reaction solution is directly purified by preparative high-performance liquid chromatography to obtain two isomers of the title compound (2-27-A: 5.73 mg , 2-27-B: 7.59mg)

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% trifluoroacetic acid)

time[min] Mobile phase A[%] Mobile phase B [%] Flow rate [mL/min] 0.00 30 90 28 2.00 30 90 28 18.00 90 10 28

The structural characterization data of 2-27-A are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.45 (d, J = 9.1Hz, 1H), 8.14 (s, 1H), 7.30 (s, 1H), 6.54 (s, 1H), 5.63 (s, 1H),5.56(q,J＝8.0Hz,1H),5.42(s,2H),5.25(d,J＝19.0Hz,1H),5.08(d,J＝19.0Hz,1H),4.13(q, J＝6.7Hz,1H),3.27–3.12(m,2H),2.51(s,3H),2.23–2.13(m,2H),1.92–1.80(m,2H),1.41(d,J＝6.8Hz ,3H),0.87(t,J=7.3Hz,3H).

ESI-MS(m/z):524.2[M+H] ⁺ .

The structural characterization data of 2-27-B are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.40 (d, J = 8.9 Hz, 1H), 8.15 (s, 1H), 7.31 (s, 1H), 6.54 (s, 1H), 5.55–5.49 ( m,1H),5.43(d,J＝2.2Hz,2H),5.18(q,J＝19.0Hz,2H),4.13(q,J＝6.6Hz,1H),3.24–3.12(m,2H), 2.51(s,3H),2.22–2.10(m,2H),1.92–1.82(m,2H),1.30(d,J＝6.7Hz,3H),0.88(t,J＝7.3Hz,3H).

ESI-MS(m/z):524.2[M+H] ⁺ .

Example 25: (R)-N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxy-2,3,9, 10,13,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazine[1,2-b]quinolin-1-yl)- 2-Hydroxypropylamine and (R)-N-((1R,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxy-2,3,9,10 ,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazine[1,2-b]quinolin-1-yl)-2 -Preparation of hydroxypropylamine

At 25°C, 3-1-A (20.0 mg, 43.9 μmol) and D-lactic acid (7.90 mg, 87.8 μmol) were dissolved in DMF (1.0 mL), and then HATU (33.4 mg, 87.8 μmol) and DIPEA ( 17.0 mg, 131.6 μmol), react at room temperature for 2 hr; the reaction solution was concentrated to remove most of the DMF, and the residue was purified by preparative high-performance liquid chromatography to obtain compound 3-26-A (15.4 mg, yield 64%).

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

Retention time: 5.3～6.2min

time[min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL/min] 0.00 30 70 28 2.00 30 70 28 18.00 90 10 28

The structural characterization data are as follows:

MS m/z(ESI):528.2[M+H] ⁺

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.52 (d, J = 9.2 Hz, 1H), 8.05 (d, J = 10.0 Hz, 1H), 7.33 (s, 1H), 6.55 (s, 1H) ,5.62–5.59(m,2H),5.43(s,2H),5.28–5.10(m,2H),4.13–4.11(m,1H),3.41–3.38(m,1H),3.28–3.22(m, 1H),2.20–2.18(m,2H),1.92–1.80(m,2H),1.40(d,J＝6.8Hz,3H),0.87(t,J＝7.2Hz,3H).

At 25°C, 3-1-B (20.0 mg, 43.9 μmol) and D-lactic acid (7.90 mg, 87.8 μmol) were dissolved in DMF (1.0 mL), and then HATU (33.4 mg, 87.8 μmol) and DIPEA ( 17.0 mg, 131.6 μmol), react at room temperature for 2 hr; the reaction solution was concentrated to remove most of the DMF, and the residue was purified by preparative high-performance liquid chromatography to obtain compound 3-26-B (4.8 mg, yield 20%).

Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

Retention time: 7.5～8.5min

time[min] Mobile phase A[%] Mobile phase B [%] Flow rate [mL/min] 0.00 30 70 28 4.00 30 70 28 20.00 90 10 28

The structural characterization data are as follows:

MS m/z(ESI):528.2[M+H] ⁺

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.48 (d, J = 8.8 Hz, 1H), 8.06 (d, J = 10.4 Hz, 1H), 7.34 (s, 1H), 6.57 (br, 1H) ,5.61–5.55(m,1H),5.48–5.39(m,2H),5.27–5.16(m,2H),4.15–4.10(m,1H),3.32–3.21(m,3H),2.23–2.16( m,2H),1.92–1.81(m,2H),1.30(d,J＝6.4Hz,3H),0.87(t,J＝7.2Hz,3H).

biological evaluation

1. Tumor cell proliferation inhibition test

1. Inhibitory effect of compounds on HT29 cell proliferation

(1) Cell plating: First, use the corresponding culture medium to culture tumor cells HT29, digest the cells with trypsin, resuspend the cells after centrifugation, count them, and adjust the cells to an appropriate concentration for plating. The sources of tumor cells are shown in Table 1.

Table 1. Source of tumor cells

cell name Tumor type source HT29 human colon cancer cells Cell Bank of Chinese Academy of Sciences

Co-incubation of the compounds of the present invention and tumor cells: After the cells adhere to the wall, remove the culture medium from the cells, add the diluted bioactive molecules (compounds of the present invention) to the wells of the above plate, and incubate for 72 hours.

In vitro cell viability detection: After the incubation, add 50 μL of Cell Counting-Lite ^TM 2.0 reagent (Vazyme/Norvizan) to each well, shake and mix well in the dark, and react for 10 minutes before testing. Microplate reader (manufacturer: BMG, Model: PHERAStar-FS) reading. Background RLU was obtained using cell-free medium (containing Cell Counting-Lite ^™ ), and vehicle RLU was obtained using cell-containing medium (containing Cell Counting-Lite ^™ ). Cell inhibition rate = 1-(sample RLU-sample RLU)/(vehicle RLU-background RLU) × 100%. Fit the curve according to the four-parameter model to calculate the half inhibitory concentration (IC50) of the compound. The test results are shown in Table 2. .

(2)Data results

Table 2. HT29 cell proliferation inhibitory activity

name IC ₅₀ (nM) Comparative compound one 11.27 Example 1 (1-1-A) 1.28 Example 1 (1-1-B) 1.54 Embodiment 3 (2-1-A) 5.03 Embodiment 3 (2-2-B) 7.52 Example 4 (2-7-A) 1.56 Example 4 (2-7-B) 5.44 Example 5 (2-12-A) 3.01 Example 5 (2-12-B) 2.34 Example 5 (2-12-C) 1.49 Example 5 (2-12-D) 3.29 Example 6 (2-17-A) 1.24 Example 6 (2-17-B) 1.59 Embodiment 7 (2-20-A) 1.29 Embodiment 7 (2-20-B) 4.03 Embodiment 8 (3-1-A) 0.16 Embodiment 8 (3-1-B) 0.71 Embodiment 9 (3-4-A) 5.55 Embodiment 9 (3-4-B) 4.98 Embodiment 10 (4-12) 4.79 Embodiment 11 (4-10) 0.52 Example 12 (5-13-A) 0.88 Example 13 (5-7-A) 3.13 Embodiment 13 (5-7-B) 4.66 Example 14 (5-16-A) 3.87 Example 20 (1-10-A) 61.60 Example 20 (1-10-B) 10.26 Embodiment 21 (4-14) 0.56

Embodiment 22 (4-15) 5.20

The test results show that the compounds of the present invention in Table 2 have a strong inhibitory effect on the proliferation of HT29 colon cancer cells.

2. Inhibitory effect of compounds on A549 cell proliferation

(1) Cell plating: First, use the corresponding culture medium to culture tumor cells A549, digest the cells with trypsin, resuspend the cells after centrifugation, count them, and adjust the cells to an appropriate concentration for plating. The sources of tumor cells are shown in Table 3.

Table 3. Source of tumor cells

cell name Tumor type source A549 human lung cancer cells Cell Bank of Chinese Academy of Sciences

Co-incubation of the compounds of the present invention and tumor cells: After the cells adhere to the wall, remove the culture medium from the cells, add the diluted bioactive molecules (compounds of the present invention) to the wells of the above plate, and incubate for 72 hours.

In vitro cell viability detection: After incubation, add 50 0L of Cell Counting-Lite ^TM 2.0 reagent (Vazyme/Norvizan) to each well, shake and mix well in the dark, and react for 10 minutes before testing. Microplate reader (Manufacturer: BMG , Model: PHERAStar-FS) reading. Background RLU was obtained using cell-free medium (containing Cell Counting-Lite ^™ ), and vehicle RLU was obtained using cell-containing medium (containing Cell Counting-Lite ^™ ). Cell inhibition rate = 1-(sample RLU-sample RLU)/(vehicle RLU-background RLU) × 100%. Fit the curve according to the four-parameter model to calculate the half inhibitory concentration (IC50) of the compound. The test results are shown in Table 4. .

(2)Data results

Table 4. A549 cell proliferation inhibitory activity

name IC ₅₀ (nM) Comparative compound one 94.18 Example 1 (1-1-A) 66.56 Example 4 (2-7-A) 6.97 Example 4 (2-7-B) 16.39 Example 5 (2-12-A) 6.96 Example 5 (2-12-B) 5.52 Example 5 (2-12-C) 6.64 Embodiment 7 (2-20-A) 8.14 Embodiment 7 (2-20-B) 23.06 Embodiment 11 (4-10) 68.48

The test results show that the compounds of the present invention in Table 4 have a significant inhibitory effect on the proliferation of A549 human lung cancer cells.

3. Inhibitory effect of compounds on NCI-H1806 cell proliferation

(1) Cell plating: First, use the corresponding culture medium to culture tumor cells HCC1806, digest the cells with trypsin, resuspend the cells after centrifugation, count them, and adjust the cells to an appropriate concentration for plating. The sources of tumor cells are shown in Table 5.

Table 5. Source of tumor cells

cell name Tumor type source HCC1806 human breast squamous cell carcinoma ATCC

Co-incubation of the compounds of the present invention and tumor cells: After the cells adhere to the wall, remove the culture medium from the cells, add the diluted bioactive molecules (compounds of the present invention) to the wells of the above plate, and incubate for 72 hours.

In vitro cell viability detection: After the incubation, add 50 μL of Cell Counting-Lite ^TM 2.0 reagent (Vazyme/Norvizan) to each well, shake and mix well in the dark, and react for 10 minutes before testing. Microplate reader (manufacturer: BMG, Model: PHERAStar-FS) reading. Background RLU was obtained using cell-free medium (containing Cell Counting-Lite ^™ ), and vehicle RLU was obtained using cell-containing medium (containing Cell Counting-Lite ^™ ). Cell inhibition rate = 1-(sample RLU-sample RLU)/(vehicle RLU-background RLU)×100%. Fit the curve according to the four-parameter model to calculate the half inhibitory concentration (IC50) of the compound. The test results are shown in Table 6 .

(2)Data results

Table 6. HCC1806 cell proliferation inhibitory activity

name IC ₅₀ (nM) Comparative compound one 3.03 Embodiment 3 (2-1-A) 1.43 Example 4 (2-7-A) 1.25 Example 5 (2-12-A) 1.40 Example 5 (2-12-B) 1.07 Example 5 (2-12-C) 2.08 Example 12 (5-13-A) 0.96 Example 16 (3-12-A) 0.16 Example 16 (3-12-B) 1.76 Example 16 (3-12-C) 1.91 Example 16 (3-12-D) 1.53 Embodiment 17 (3-7-B) 1.94 Example 18 (3-17-A) 1.70 Example 18 (3-17-B) 2.26 Example 19 (5-22-A) 2.57 Example 19 (5-22-B) 3.71 Example 24 (2-27-A) 4.55 Example 24 (2-27-B) 3.93 Example 25 (3-26-A) 1.27

Example 25 (3-26-B) 4.93

The test results show that the compounds of the present invention in Table 6 have a significant inhibitory effect on the proliferation of HCC1806 human breast squamous cancer cells.

4. Inhibitory effect of compounds on SKOV-3 cell proliferation

(1) Cell plating: First, use the corresponding culture medium to culture tumor cells SKOV-3, digest the cells with trypsin, resuspend the cells after centrifugation, count them, and adjust the cells to an appropriate concentration for plating. The sources of tumor cells are shown in Table 7.

Table 7. Source of tumor cells

cell name Tumor type source SKOV-3 human ovarian cancer cells Nanjing Kebai Biotechnology

Co-incubation of the compounds of the present invention and tumor cells: After the cells adhere to the wall, remove the culture medium from the cells, add the diluted bioactive molecules (compounds of the present invention) to the above-mentioned plate wells, and incubate for 72 hours.

In vitro cell viability detection: After the incubation, add 50μ0 of Cell Counting-Lite ^TM 2.0 reagent (Vazyme/Novizan) to each well, shake and mix well in the dark, and react for 10 minutes before testing. Microplate reader (manufacturer: BMG, Model: PHERAStar-FS) reading. Background RLU was obtained using cell-free medium (containing Cell Counting-Lite ^™ ), and vehicle RLU was obtained using cell-containing medium (containing Cell Counting-Lite ^™ ). Cell inhibition rate = 1-(sample RLU-sample RLU)/(vehicle RLU-background RLU)×100%, fit the curve according to the four-parameter model, and calculate the half inhibitory concentration (IC50) of the compound. The test results are shown in Table 8 .

(2)Data results

Table 8. SKOV-3 cell proliferation inhibitory activity

name IC ₅₀ (nM) Comparative compound one 17.14 Example 1 (1-1-A) 7.81 Embodiment 3 (2-1-A) 7.12 Example 4 (2-7-A) 8.64 Example 4 (2-7-B) 9.47 Example 5 (2-12-A) 6.85 Example 5 (2-12-B) 4.99 Example 5 (2-12-C) 6.52 Embodiment 7 (2-20-A) 6.79 Example 12 (5-13-A) 2.09 Embodiment 21 (4-14) 2.16

The test results show that the compounds of the present invention in Table 8 have a significant inhibitory effect on the proliferation of SKOV-3 human ovarian cancer cells.

5. Inhibitory effect of compounds on NCI-H358 cell proliferation

(1) Cell plating: First, use the corresponding culture medium to culture tumor cells NCI-H358, digest the cells with trypsin, resuspend the cells after centrifugation, count them, and adjust the cells to an appropriate concentration for plating. The sources of tumor cells are shown in Table 9.

Table 9. Source of tumor cells

cell name Tumor type source NCI-H358 human non-small cell lung cancer cells Nanjing Kebai Biotechnology

Co-incubation of the compounds of the present invention and tumor cells: After the cells adhere to the wall, remove the culture medium from the cells, add the diluted bioactive molecules (compounds of the present invention) to the wells of the above plate, and incubate for 72 hours.

In vitro cell viability detection: After the incubation, add 50μ0 of Cell Counting-Lite ^TM 2.0 reagent (Vazyme/Novizan) to each well, shake and mix well in the dark, and react for 10 minutes before testing. Microplate reader (manufacturer: BMG, Model: PHERAStar-FS) reading. Background RLU was obtained using cell-free medium (containing Cell Counting-Lite ^™ ), and vehicle RLU was obtained using cell-containing medium (containing Cell Counting-Lite ^™ ). Cell inhibition rate = 1-(sample RLU-sample RLU)/(vehicle RLU-background RLU)×100%, fit the curve according to the four-parameter model, and calculate the half inhibitory concentration (IC50) of the compound. The test results are shown in Table 10 .

(2)Data results

Table 10. NCI-H358 cell proliferation inhibitory activity

name IC ₅₀ (nM) Comparative compound one 58.84 Example 1 (1-1-A) 65.22 Example 2 (1-7-A) 11.38 Example 2 (1-7-B) 51.52 Embodiment 3 (2-1-A) 68.62 Example 4 (2-7-A) 35.42 Example 4 (2-7-B) 51.06 Example 5 (2-12-A) 17.65 Example 5 (2-12-B) 15.76 Example 5 (2-12-C) 23.08 Embodiment 7 (2-20-A) 18.56 Example 12 (5-13-A) 7.01 Example 16 (3-12-A) 15.45 Example 16 (3-12-B) 19.77 Example 16 (3-12-C) 28.29 Example 16 (3-12-D) 20.75 Embodiment 17 (3-7-A) 49.38 Embodiment 17 (3-7-B) 22.22

Example 18 (3-17-A) 13.34 Example 18 (3-17-B) 37.34 Example 19 (5-22-A) 29.94 Example 19 (5-22-B) 61.36 Embodiment 21 (4-14) 11.98 Embodiment 22 (4-15) 17.76 Example 24 (2-27-A) 56.46 Example 24 (2-27-B) 69.12 Example 25 (3-26-A) 31.64

The test results show that the compounds of the present invention in Table 10 have a significant inhibitory effect on the proliferation of NCI-H358 human non-small cell lung cancer cells.

6. Inhibitory effect of compounds on NCI-N87 cell proliferation

(1) Cell plating: First, use the corresponding culture medium to culture tumor cells NCI-N87, digest the cells with trypsin, centrifuge and resuspend the cells for counting, adjust the cells to an appropriate concentration for plating. The sources of tumor cells are shown in Table 11.

Table 11. Source of tumor cells

cell name Tumor type source NCI-N87 human gastric cancer cells ATCC

Co-incubation of the compounds of the present invention and tumor cells: After the cells adhere to the wall, remove the culture medium from the cells, add the diluted bioactive molecules (compounds of the present invention) to the wells of the above plate, and incubate for 72 hours.

In vitro cell viability detection: After incubation, add 50 μL of Cell Counting-Lite ^TM 2.0 reagent (Vazyme/Novizan) to each well, shake and mix well in the dark, and react for 10 minutes before testing. Microplate reader (manufacturer: BMG, Model: PHERAStar-FS) reading. Background RLU was obtained using cell-free medium (containing Cell Counting-Lite ^™ ), and vehicle RLU was obtained using cell-containing medium (containing Cell Counting-Lite ^™ ). Cell inhibition rate = 1-(sample RLU-sample RLU)/(vehicle RLU-background RLU)×100%. Fit the curve according to the four-parameter model to calculate the half inhibitory concentration (IC50) of the compound. The test results are shown in Table 12 .

(2)Data results

Table 12. NCI-N87 cell proliferation inhibitory activity

name IC ₅₀ (nM) Comparative compound one 7.18 Example 1 (1-1-A) 4.58 Example 2 (1-7-A) 2.70 Example 2 (1-7-B) 5.48 Example 4 (2-7-A) 3.76 Example 5 (2-12-A) 4.91

Example 5 (2-12-B) 4.97 Example 5 (2-12-C) 4.51 Embodiment 7 (2-20-A) 4.54 Embodiment 8 (3-1-A) 0.37 Embodiment 8 (3-1-B) 1.34 Embodiment 9 (3-4-A) 4.38 Embodiment 9 (3-4-B) 6.51 Embodiment 11 (4-10) 2.29 Example 12 (5-13-A) 2.33 Example 13 (5-7-A) 6.03 Embodiment 13 (5-7-B) 7.31 Example 16 (3-12-A) 0.44 Example 16 (3-12-B) 3.45 Example 16 (3-12-C) 3.99 Example 16 (3-12-D) 3.03 Embodiment 17 (3-7-A) 5.32 Embodiment 17 (3-7-B) 4.54 Example 18 (3-17-A) 3.01 Example 18 (3-17-B) 4.19 Example 19 (5-22-A) 3.35 Example 19 (5-22-B) 5.51 Example 24 (2-27-A) 8.32 Example 24 (2-27-B) 14.67 Example 25 (3-26-A) 1.23 Example 25 (3-26-B) 25.34

The test results show that the compounds of the present invention in Table 12 have a significant inhibitory effect on the proliferation of NCI-N87 human gastric cancer cells.

7. Inhibitory effect of compounds on HeLa cell proliferation

(1) Cell plating: First, use the corresponding culture medium to culture tumor cells HeLa, digest the cells with trypsin, centrifuge and resuspend the cells for counting, adjust the cells to an appropriate concentration for plating. The sources of tumor cells are shown in Table 13.

Table 13. Source of tumor cells

cell name Tumor type source Hela human cervical cancer cells Nanjing Kebai Biotechnology

Co-incubation of the compounds of the present invention and tumor cells: After the cells adhere to the wall, remove the culture medium from the cells, add the diluted bioactive molecules (compounds of the present invention) to the above-mentioned plate wells, and incubate for 72 hours.

In vitro cell viability detection: After incubation, add 50 μL of Cell Counting-Lite ^TM 2.0 reagent (Vazyme/Novizan) to each well, shake and mix well in the dark, and react for 10 minutes before testing. Microplate reader (manufacturer: BMG, Model: PHERAStar-FS) reading. Background RLU was obtained using cell-free medium (containing Cell Counting-Lite ^™ ), and vehicle RLU was obtained using cell-containing medium (containing Cell Counting-Lite ^™ ). Cell inhibition rate = 1-(sample RLU-sample RLU)/(vehicle RLU-background RLU)×100%, fit the curve according to the four-parameter model, and calculate the half inhibitory concentration (IC50) of the compound. The test results are shown in Table 14 .

(2)Data results

Table 14. Hela cell proliferation inhibitory activity

name IC ₅₀ (nM) Comparative compound one 17.57 Example 1 (1-1-A) 4.57 Example 1 (1-1-B) 3.65 Embodiment 3 (2-1-A) 16.98 Example 4 (2-7-A) 13.77 Example 5 (2-12-A) 13.25 Example 5 (2-12-B) 16.68 Embodiment 7 (2-20-A) 11.17 Embodiment 11 (4-10) 3.04

The test results show that the compounds of the present invention in Table 14 have a significant inhibitory effect on the proliferation of HeLa human cervical cancer cells.

8. Inhibitory effect of compounds on HCC70 cell proliferation

(1) Cell plating: First, use the corresponding culture medium to culture tumor cells HCC70, digest the cells with trypsin, resuspend the cells after centrifugation, count them, and adjust the cells to an appropriate concentration for plating. The sources of tumor cells are shown in Table 15.

Table 15. Source of tumor cells

cell name Tumor type source HCC70 human breast cancer cells Nanjing Kebai Biotechnology

Co-incubation of the compounds of the present invention and tumor cells: After the cells adhere to the wall, remove the culture medium from the cells, add the diluted bioactive molecules (compounds of the present invention) to the above-mentioned plate wells, and incubate for 72 hours.

In vitro cell viability detection: After the incubation, add 50μ0 of Cell Counting-Lite ^TM 2.0 reagent (Vazyme/Novizan) to each well, shake and mix well in the dark, and react for 10 minutes before testing. Microplate reader (manufacturer: BMG, Model: PHERAStar-FS) reading. Background RLU was obtained using cell-free medium (containing Cell Counting-Lite ^™ ), and vehicle RLU was obtained using cell-containing medium (containing Cell Counting-Lite ^™ ). Cell inhibition rate = 1-(sample RLU-sample RLU)/(vehicle RLU-background RLU)×100%, fit the curve according to the four-parameter model, and calculate the half inhibitory concentration (IC50) of the compound. The test results are shown in Table 16 .

(2)Data results

Table 16. HCC70 cell proliferation inhibitory activity

name IC ₅₀ (nM) Comparative compound one 226.10 Example 1 (1-1-A) 224.85 Example 4 (2-7-A) 150.70 Example 4 (2-7-B) 192.20 Example 5 (2-12-A) 170.93 Example 5 (2-12-B) 176.16 Example 5 (2-12-C) 210.68 Embodiment 7 (2-20-A) 104.99 Example 12 (5-13-A) 36.65

The test results show that the compounds of the present invention in Table 16 have a significant inhibitory effect on the proliferation of HCC70 human breast cancer cells.

9. Inhibitory effect of compounds on MDA-MB-231 cell proliferation

(1) Cell plating: First, use the corresponding culture medium to culture tumor cells MDA-MB-231, digest the cells with trypsin, resuspend the cells after centrifugation, count them, and adjust the cells to an appropriate concentration for plating. The sources of tumor cells are shown in Table 17.

Table 17. Source of tumor cells

cell name Tumor type source MDA-MB-231 human breast cancer cells Nanjing Kebai Biotechnology

Co-incubation of the compounds of the present invention and tumor cells: After the cells adhere to the wall, remove the culture medium from the cells, add the diluted bioactive molecules (compounds of the present invention) to the above-mentioned plate wells, and incubate for 72 hours.

In vitro cell viability detection: After incubation, add 50) m of Cell Counting-Lite ^TM 2.0 reagent (Vazyme/Norvizan) to each well, shake and mix well in the dark, and react for 10 minutes before testing. Microplate reader (manufacturer: BMG, model: PHERAStar-FS) reading. Background RLU was obtained using cell-free medium (containing Cell Counting-Lite ^™ ), and vehicle RLU was obtained using cell-containing medium (containing Cell Counting-Lite ^™ ). Cell inhibition rate = 1-(sample RLU-sample RLU)/(vehicle RLU-background RLU)×100%, fit the curve according to the four-parameter model, and calculate the half inhibitory concentration (IC50) of the compound. The test results are shown in Table 18 .

(2)Data results

Table 18. MDA-MB-231 cell proliferation inhibitory activity

name IC ₅₀ (nM) Comparative compound one 381.70 Example 1 (1-1-A) 363.60

Embodiment 3 (2-1-A) 299.70 Example 4 (2-7-A) 123.50 Example 5 (2-12-A) 123.50 Embodiment 9 (3-4-A) 284.80 Example 12 (5-13-A) 332.60 Embodiment 13 (5-7-B) 172.40 Example 20 (1-10-A) 97.57 Example 20 (1-10-B) 27.24 Embodiment 22 (4-15) 175.40

The test results show that the compounds of the present invention in Table 18 have a significant inhibitory effect on the proliferation of MDA-MB-231 human breast cancer cells.

10. Inhibitory effect of compounds on Jeko-1 cell proliferation

(1) Cell plating: First, use the corresponding culture medium to culture tumor cells Jeko-1, centrifuge the cells, resuspend and count the cells, and finally adjust the cells to an appropriate concentration for plating. The sources of tumor cells are shown in Table 19.

Table 19. Source of tumor cells

cell name Tumor type source Jeko-1 human mantle cell lymphoma cells ATCC

Co-incubation of the compounds of the present invention and tumor cells: After the cells are plated, the diluted bioactive molecules (compounds of the present invention) are added to the above-mentioned plate wells according to a certain dilution ratio, and incubated for 72 hours.

In vitro cell viability detection: After incubation, add 50 0% Cell Counting-Lite ^TM 2.0 reagent (Vazyme/Norvizan) to each well, shake and mix well in the dark, and react for 10 minutes before testing. Microplate reader (manufacturer: BMG, model: PHERAStar-FS) reading. Background RLU was obtained using cell-free medium (containing Cell Counting-Lite ^™ ), and vehicle RLU was obtained using cell-containing medium (containing Cell Counting-Lite ^™ ). Cell inhibition rate = 1-(sample RLU-sample RLU)/(vehicle RLU-background RLU)×100%, fit the curve according to the four-parameter model, and calculate the half inhibitory concentration (IC50) of the compound. The test results are shown in Table 20 .

(2)Data results

Table 20. Jeko-1 cell proliferation inhibitory activity

name IC ₅₀ (nM) Comparative compound one 1.25 Example 1 (1-1-A) 1.33 Example 2 (1-7-A) 0.15 Example 2 (1-7-B) 0.66 Example 4 (2-7-A) 0.28 Embodiment 9 (3-4-A) 0.27

Embodiment 9 (3-4-B) 0.15 Embodiment 11 (4-10) 0.45 Example 13 (5-7-A) 0.16 Embodiment 13 (5-7-B) 0.18 Embodiment 21 (4-14) 0.34 Embodiment 22 (4-15) 0.11

The test results show that the compounds of the present invention in Table 20 have a significant inhibitory effect on the proliferation of Jeko-1 human mantle cell lymphoma cells.

11. Inhibitory effect of compounds on MDA-MB-453 cell proliferation

(1) Cell plating: First, use the corresponding culture medium to culture tumor cells MDA-MB-453, digest the cells with trypsin, resuspend the cells after centrifugation, count them, and adjust the cells to an appropriate concentration for plating. The sources of tumor cells are shown in Table 21.

Table 21. Source of tumor cells

cell name Tumor type source MDA-MB-453 human breast cancer cells Connaught

Co-incubation of the compounds of the present invention and tumor cells: After the cells adhere to the wall, remove the culture medium from the cells, add the diluted bioactive molecules (compounds of the present invention) to the above-mentioned plate wells, and incubate for 72 hours.

In vitro cell viability detection: After incubation, add 50 0% Cell Counting-Lite ^TM 2.0 reagent (Vazyme/Norvizan) to each well, shake and mix well in the dark, and react for 10 minutes before testing. Microplate reader (manufacturer: BMG, model: PHERAStar-FS) reading. Background RLU was obtained using cell-free medium (containing Cell Counting-Lite ^™ ), and vehicle RLU was obtained using cell-containing medium (containing Cell Counting-Lite ^™ ). Cell inhibition rate = 1-(sample RLU-sample RLU)/(vehicle RLU-background RLU)×100%, fit the curve according to the four-parameter model, and calculate the half inhibitory concentration (IC ₅₀ ) of the compound. The test results are shown in Table 22 Show.

(2)Data results

Table 22. MDA-MB-453 cell proliferation inhibitory activity

name IC ₅₀ (nM) Comparative compound one 6.99 Example 16 (3-12-A) 0.47 Example 16 (3-12-B) 4.32 Example 16 (3-12-C) 5.52 Example 16 (3-12-D) 3.98 Embodiment 17 (3-7-A) 6.23 Embodiment 17 (3-7-B) 4.87 Example 18 (3-17-A) 3.32 Example 18 (3-17-B) 6.16

Example 19 (5-22-A) 7.34 Example 19 (5-22-B) 5.67 Example 24 (2-27-A) 11.83 Example 25 (3-26-A) 3.95

The test results show that the compounds of the present invention in Table 22 have a significant inhibitory effect on the proliferation of MDA-MB-453 human breast cancer cells.

The structures of Comparative Compound 1 and Comparative Compound 2 are as follows:

2. Antibody coupling test

The coupling preparation of ADC D-L-15 sample is as follows:

Take 1.036mL hIgG antibody (anti-chicken lysozyme antibody, 19.3mg/mL), dilute it with 0.1M disodium edetate solution (pH 7.6), then adjust the pH to 7.6 with 1M Na ₂ HPO ₄ solution, and add 2.4 times Mix the amount of substance with 10mM TCEP (tris(2-carboxyethyl)phosphine) solution (pH 7.6) and leave it at room temperature for 90 minutes. Add 5 times the amount of compound DL-15 dissolved in dimethyl sulfoxide to the above solution system, mix well, and let stand at room temperature for 2 hours. After completion, use a NAP-5 gel column (Cytiva) to replace the buffer with pH. 6.0 of 10mM histidine buffer solution, then add sucrose and Tween 20, mix well, and obtain antibody drug conjugate ADC DL-15 (1.77mL, 8.60mg/mL).

The molecular weight of ADC D-L-15 was measured using LC-MS, and the drug/antibody ratio DAR value was calculated to be 4.11, as shown in Table 23 and Table 24.

Table 23: Measured molecular weight of ADC D-L-15

Table 24: DAR values for ADC D-L-15

Chromatography conditions:

Liquid chromatography column: Thermo MAbPac RP 3.0*100mm;

Mobile phase A: 0.1% FA/H ₂ O; mobile phase B: 0.1% FA/ACN;

Flow rate: 0.25mL/min; sample chamber temperature: 8℃; column temperature: 60℃; injection volume: 2μL;

time (minutes) 2 20 twenty two 25 26 30 Mobile phase A (volume %) 75 60 5 5 75 75 Mobile phase B (volume%) 25 40 95 95 25 25

Mass spectrometry conditions:

Mass spectrometer model: AB Sciex Triple TOF 5600+;

GS1 35; GS2 35; CUR 30; TEM 350; ISVF 5500; DP 250; CE 10; Accumulation time 0.5s; m/z 600-4000; Time bins to sum 40.

The coupling preparation of ADC 3-4-04-A sample is as follows:

Take 0.518mL hIgG antibody (anti-chicken lysozyme antibody, 19.3mg/mL), dilute it with 0.1M disodium edetate solution (pH 7.6), then adjust the pH to 7.6 with 1M Na ₂ HPO ₄ solution, and add 5.5 times Mix the amount of substance with 10mM TCEP (tris(2-carboxyethyl)phosphine) solution (pH 7.6) and leave it at room temperature for 90 minutes. Add 10 times the amount of compound 3-4-04-A dissolved in dimethyl sulfoxide to the above solution system, mix well, and let stand at room temperature for 2 hours. After completion, use NAP-5 gel column (Cytiva) to remove the buffer. The solution was replaced with a 10mM histidine buffer solution with pH 6.0, and then sucrose and Tween 20 were added and mixed to obtain antibody drug conjugate ADC 3-4-04-A (1.50mL, 5.60mg/mL).

The molecular weight of ADC 3-4-04-A was measured using LC-MS, and the drug/antibody ratio DAR value was calculated to be 7.47, as shown in Table 25 and Table 26. The chromatographic measurement conditions were the same as ADC D-L-15.

Table 25: Measured molecular weight of ADC 3-4-04-A

Table 26: DAR values for ADC 3-4-04-A

The coupling preparation of ADC 3-4-04-B sample is as follows:

Take 0.518mL hIgG antibody (anti-chicken lysozyme antibody, 19.3mg/mL), dilute it with 0.1M disodium edetate solution (pH 7.6), then adjust the pH to 7.6 with 1M Na ₂ HPO ₄ solution, and add 5.5 times Mix the amount of substance with 10mM TCEP (tris(2-carboxyethyl)phosphine) solution (pH 7.6) and leave it at room temperature for 90 minutes. Add 10 times the amount of compound 3-4-04-B dissolved in dimethyl sulfoxide to the above solution system, mix well, and let stand at room temperature for 2 hours. After completion, use NAP-5 gel column (Cytiva) to remove the buffer. The solution was replaced with a 10mM histidine buffer solution with pH 6.0, and then sucrose and Tween 20 were added and mixed to obtain antibody drug conjugate ADC 3-4-04-B (1.50mL, 5.60mg/mL).

The molecular weight of ADC 3-4-04-B was measured using LC-MS, and the drug/antibody ratio DAR value was calculated to be 8.04, as shown in Table 27 and Table 28. The chromatographic measurement conditions were the same as ADC D-L-15.

Table 27: Measured molecular weight of ADC 3-4-04-B

Table 28: DAR values for ADC 3-4-04-B

The above experiments prove that the cytotoxic drug-linker compounds of the present invention can be successfully coupled with antibodies to obtain antibody drug conjugates.

Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all teachings that have been disclosed, and these changes are within the scope of the present invention. . The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims (14)

1. A compound or pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotopic labels, metabolites and prodrugs thereof, wherein said compound has the structure shown below:

   wherein,

   R ₁ and R is ₂ Each independently selected from hydrogen, halogen, C _1-6 Alkyl, C _1-6 Alkoxy, C _1-6 Haloalkyl, hydroxy, cyano and C _3-6 Cycloalkyl; alternatively, R ₁ And R is ₂ Is connected with adjacent carbon atoms to form 5-6 membered oxygen-containing heterocycle;

   R ₃ is hydrogen or with R ₁ The ortho carbon atoms are linked to form a six membered carbocyclic ring;

   a is selected fromOne of the following;

   R ₄ selected from hydrogen, C _1-6 Alkyl, C _1-6 Haloalkyl, C _1-6 Alkoxyalkyl, C _3-6 Cycloalkyl and 3-6 membered heterocyclyl;

   R ₅ and R is ₆ Each independently selected from hydrogen, C _1-6 Alkyl, C _1-6 HaloalkanesRadical, C _1-6 Alkylaminoalkyl, C _1-6 Alkoxyalkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _3-6 Cycloalkyl, 3-6 membered heterocyclyl, aryl and heteroaryl; or R is ₅ And R is ₆ Is linked to adjacent carbon atoms to form a 3-6 membered carbocyclic or heterocyclic ring;

   m=1 or 2.
2. The compound of claim 1, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitroxide, isotopic label, metabolite, and prodrug thereof, wherein the compound has the structure of formula (I):

   in the above formula (I), R ^x Selected from hydrogen, C _1-6 Alkyl, C _1-6 Haloalkyl, C _1-6 Alkoxyalkyl, C _3-6 Cycloalkyl and 3-6 membered heterocyclyl;

   R ^y and R is ^z Are not hydrogen at the same time and are independently selected from hydrogen, C _1-6 Alkyl, C _1-6 Haloalkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _1-6 Alkylaminoalkyl, C _1-6 Alkoxyalkyl groups, 3-6 membered heterocyclylalkyl groups, and 3-6 membered heterocyclyl groups;

   preferably, R ^x Selected from hydrogen and C _1-6 An alkyl group; preferably, R ^x Is hydrogen;

   preferably, R ^y And R is ^z Are not hydrogen at the same time and are independently selected from hydrogen,Dimethylaminomethylene, morpholinylmethyleneAnd methoxymethylene;

   preferably, R ^y Is hydrogen, R ^z Selected from the group consisting ofDimethylaminomethylene, morpholinomethylene and methoxymethylene.
3. The compound of claim 1, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitroxide, isotopic label, metabolite, and prodrug thereof, wherein the compound has the structure of formula (II):

   In the above formula (II), A' is selected fromOne of the following;

   R ^x’ selected from hydrogen, C _1-6 Alkyl, C _1-6 Haloalkyl, C _1-6 Alkoxyalkyl, C _3-6 Cycloalkyl and 3-6 membered heterocyclyl;

   R ^y’ and R is ^z’ Independently selected from hydrogen, C _1-6 Alkyl, C _1-6 Haloalkyl, C _1-6 Alkoxyalkyl, C _1-6 Alkylaminoalkyl, C _1-6 Alkoxyalkyl, C _3-6 Cycloalkyl, 3-6 membered heterocyclyl, 3-6 membered heterocyclylalkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, aryl and heteroaryl, or R ^y’ And R is ^z’ Is connected with adjacent carbon atoms to form a 3-6 membered ring;

   preferably, R ^x’ Selected from hydrogenAnd C _1-6 An alkyl group;

   preferably, R ^y’ And R is ^z’ Independently selected from hydrogen, C _1-6 Alkyl, C _1-6 Alkoxyalkyl, C _1-6 Alkylaminoalkyl, C _3-6 Cycloalkyl and C _2-6 Alkenyl, or R ^y’ And R is ^z’ Is connected with adjacent carbon atoms to form 3-6 membered cycloalkyl;

   preferably, R ^y’ Selected from hydrogen and C _1-6 Alkyl, R ^z’ Selected from hydrogen, C _1-6 Alkyl and C _3-6 Cycloalkyl, or R ^y’ And R is ^z’ Is connected with adjacent carbon atoms to form 3-6 membered cycloalkyl.
4. The compound of claim 1, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitroxide, isotopic label, metabolite, and prodrug thereof, wherein the compound has the structure of formula (III):

   in the above formula (III), A' is selected fromOne of the following;

   R ^x” selected from hydrogen, C _1-6 Alkyl, C _1-6 Haloalkyl, C _1-6 Alkoxyalkyl, C _3-6 Cycloalkyl and 3-6 membered heterocyclyl;

   R ^y” and R is ^z” Independently selected from hydrogen, C _1-6 Alkyl, C _1-6 Haloalkyl, C _1-6 Alkoxyalkyl, C _1-6 Alkylaminoalkyl group、C _1-6 Alkoxyalkyl, C _3-6 Cycloalkyl, 3-6 membered heterocyclyl, 3-6 membered heterocyclylalkyl, 4-6 membered heterocyclyl, C _{2- 6} Alkenyl, C _2-6 Alkynyl, aryl and heteroaryl, or R ^y” And R is ^z” Is connected with adjacent carbon atoms to form a 3-6 membered ring;

   preferably, R ^x” Is hydrogen;

   preferably, R ^y” And R is ^z” Each independently selected from hydrogen, C _1-6 Alkyl, C _1-6 Haloalkyl, C _1-6 Alkoxyalkyl, C _1-6 Alkylaminoalkyl, C _3-6 Cycloalkyl and vinyl, or R ^y” And R is ^z” Is connected with adjacent carbon atoms to form a 3-6 membered ring;

   preferably, R ^y” Is hydrogen, R ^z” Selected from hydrogen, C _1-6 Alkyl, C _3-6 Cycloalkyl and vinyl, or R ^y” And R is ^z” Is linked to adjacent carbon atoms to form a 3-6 membered carbocyclic ring.
5. The compound of claim 1, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitroxide, isotopic label, metabolite, and prodrug thereof, wherein the compound has the structure of formula (IV):

   in the above formula (IV), the amino acid sequence of the compound,

   R ^a and R is ^b Independently selected from hydrogen, halogen, C _1-6 Alkyl, C _1-6 Hydroxyalkyl, C _1-6 Alkoxyalkyl, C _1-6 Alkoxy, C _1-6 Haloalkyl, hydroxy, and cyano; or R is ^a And R is ^b Is connected with adjacent carbon atoms to form 5-6 membered oxygen-containing heterocycle;

   R ^c and R is ^d Independently selected from hydrogen, C _1-6 Alkyl, C _1-6 Haloalkyl, C _1-6 Alkoxyalkyl, C _1-6 Alkylaminoalkyl, C _3-6 Cycloalkyl, 3-6 membered heterocyclyl, 3-6 membered heterocyclylalkyl, C _2-6 Alkenyl and C _2-6 Alkynyl, or R ^c And R is ^d A carbocycle or heterocycle linked to an adjacent carbon atom in 3-6 membered ring;

   R ^e selected from hydrogen, C _1-6 Alkyl, C _3-6 Cycloalkyl, C _1-6 Haloalkyl, C _1-6 Alkoxyalkyl and C ₂ -C ₅ A heterocyclic group;

   q=0 or 1;

   when q=0, R ^c And R is ^d Are not hydrogen at the same time;

   preferably, R ^a And R is ^b Independently selected from hydrogen, halogen and C _1-6 Alkyl, or R ^a And R is ^b Is connected with adjacent carbon atoms to form 5-6 membered oxygen-containing heterocycle;

   preferably, R ^a And R is ^b Independently selected from hydrogen, fluorine, chlorine and methyl, or R ^a And R is ^b Together with the benzene ring to which it is attached form

   Wherein Z is selected from the group consisting of-CH ₂ -、-CD ₂ -、-CH ₂ CH ₂ -and-CF ₂ -；

   Preferably, R ^a Is methyl, R ^b Is fluorine, or R ^a And R is ^b Together with the benzene ring to which it is attached form

   Preferably, R ^c And R is ^d Independently selected from hydrogen,C _1-6 Alkoxyalkyl and C _1-6 Alkylaminoalkyl, or R ^c And R is ^d Is connected with adjacent carbon atoms to form a 3-6 membered carbocycle;

   preferably, R ^c Is hydrogen, R ^d Selected from hydrogen,Methoxyethyl and cyclopropyl, or R ^c And R is ^d Is connected with adjacent carbon atoms to form a 3-6 membered carbocycle;

   Preferably, R ^e Selected from hydrogen and C _1-6 An alkyl group.
6. The compound of claim 1, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitroxide, isotopic label, metabolite, and prodrug thereof, wherein the compound has the structure of formula (V):

   in the above formula (V), R is selected from C _3-6 Cycloalkyl and C _1-6 An alkoxy group;

   a' "is selected fromOne of the following;

   R ^x”’ selected from hydrogen, C _1-6 Alkyl, C _1-6 Haloalkyl, C _1-6 Alkoxyalkyl, C ₃ - ₆ Cycloalkyl and 3-6 membered heterocyclyl (e.g., 3-6 membered carbocycle or 3-6 membered heterocycle);

   R ^y”’ and R is ^z”’ Independently selected from hydrogen, C _1-6 Alkyl, C _1-6 Haloalkyl, C _1-6 Alkoxyalkyl, C _1-6 Alkylaminoalkyl, C _3-6 Cycloalkyl, 3-6 membered heterocyclyl, 3-6 membered heterocyclylalkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, aryl and heteroaryl, or R ^y”’ And R is ^z”’ Is connected with adjacent carbon atoms to form a 3-6 membered ring;

   preferably, R is selected from methyl, methoxy and cyclopropyl; preferably, R ^y”’ And R is ^z”’ Each independently selected from hydrogen, C _1-6 Alkyl, C _1-6 Haloalkyl, C _1-6 Alkoxyalkyl, C _1-6 Alkylaminoalkyl, C _3-6 Cycloalkyl and vinyl, or R ^y”’ And R is ^z”’ Is connected with adjacent carbon atoms to form a 3-6 membered carbocycle;

   preferably, R ^y”’ And R is ^z”’ Are all hydrogen, or R ^y”’ And R is ^z”’ Is connected with adjacent carbon atoms to form a 3-6 membered carbocycle;

   Preferably, R ^x”’ Selected from hydrogen and C _1-6 An alkyl group;

   preferably, R ^x”’ Is hydrogen.
7. The compound of claim 1, or pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitroxides, isotopic labels, metabolites and prodrugs thereof, said compound having the structure shown below:
8. a compound of formula (VI) or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitroxide, isotopic label, metabolite, and prodrug thereof, wherein the compound has the structure shown below:

   M-L-E-D

   (VI)

   Wherein,

   m is a linker moiety to an antibody or antigen binding fragment thereof;

   l is a linker between the linker M and E;

   e is a structural fragment linking L and D;

   d is a structural fragment of a cytotoxic drug;

   preferably, M is selected from the following structures:

   preferably, M is selected from the following structures:

   preferably, L is selected from divalent structures consisting of one or more of: c (C) _1-6 Alkylene, -N (R') -, carbonyl, -O-, val, cit, phe, lys, D-Val, leu, gly, ala, asn, val-Cit, val-Ala, val-Lys (Ac), phe-Lys (Ac), D-Val-Leu-Lys, gly-Gly-Arg, ala-Ala-Asn, ala-Ala-Ala, val-Lys-Ala, gly-Gly-Gly, gly-Gly-Phe-Gly, gly-Gly-Gly-Gly,

   Wherein R' represents hydrogen, C _1-6 Alkyl or containing- (CH) ₂ CH ₂ O) _r -an alkyl group; r is selected from integers from 1 to 10; s is selected from integers of 1-10;

   preferably, L is selected from the following structures:

   preferably, L is selected from the following structures:

   preferably E is selected from single bond, -NH-CH ₂ -、

   Preferably E is-NH-CH ₂ -；

   Preferably, the cytotoxic drug is selected from the group consisting of-compounds according to any one of claims 1-6;

   preferably, the cytotoxic drug is selected from compounds 1-1 to 1-15 of claim 7; 2-1 to 2-27;3-1 to 3-26;4-1 to 4-15; or 5-1 to 5-36;

   preferably, D is selected from the structures of any one of claims 1-6 after dehydrogenation of the compound;

   preferably, D is selected from compounds 1-1 to 1-15 of claim 7; 2-1 to 2-27;3-1 to 3-26;4-1 to 4-15; or 5-1 to 5-36 after dehydrogenation;

   preferably, D is selected from the following structures:

   preferably, D is preferably selected from the following structures:
9. a compound according to claim 8, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitroxide, isotopic label, metabolite, and prodrug thereof, said compound having the structure shown below:

   preferably, the compound is selected from:
10. a pharmaceutical composition comprising a compound according to any one of claims 1 to 9, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitroxide, isotopic label, metabolite or prodrug thereof, and one or more pharmaceutically acceptable carriers.
11. A kit product comprising:

    a) At least one compound according to any one of claims 1-9 or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, oxynitride, isotopic label, metabolite or prodrug thereof, or a pharmaceutical composition according to claim 10, as a first therapeutic agent;

    b) Optionally at least one other therapeutic agent as a second therapeutic agent, or a pharmaceutical composition comprising the other therapeutic agent as a second pharmaceutical composition; and

    c) Optionally package and/or instructions.
12. Use of a compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitroxide, isotopic label, metabolite and prodrug thereof, a pharmaceutical composition according to claim 10 or a kit product according to claim 11 for the manufacture of a medicament for the treatment of a disease in terms of abnormal proliferation of cells.
13. A method of treating a disease in terms of abnormal proliferation of cells comprising the steps of: a therapeutically effective amount of a compound of any one of claims 1-9, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, oxynitride, isotopic label, metabolite, and prodrug thereof, or a pharmaceutical composition of claim 10, for administration to a subject in need thereof.
14. A compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitroxide, isotopic label, metabolite and prodrug thereof, a pharmaceutical composition according to claim 10 or a kit product according to claim 11 for use in the treatment of a disease in terms of abnormal proliferation of cells.